George Palade’s pioneering electron microscopical studies of the pancreatic acinar cell revealed the intracellular secretory pathway from the rough endoplasmic reticulum at the base of the cell to the zymogen granules in the apical region. Palade also described for the first time the final stage of exocytotic enzyme secretion into the acinar lumen. The contemporary studies of the mechanism by which secretion is acutely controlled, and how the pancreas is destroyed in the disease acute pancreatitis, rely on monitoring molecular events in the various identified pancreatic cell types in the living pancreas. These studies have been carried out with the help of high-resolution fluorescence recordings, often in conjunction with patch clamp current measurements. In such studies we have gained much detailed information about the regulatory events in the exocrine pancreas in health as well as disease, and new therapeutic opportunities have been revealed. This was the central theme for his Nobel Prize Lecture.2 in his published Nobel Prize Lecture.1 In this paper,2 I had shown that the two physiological stimulants of exocrine pancreatic secretion, namely acetylcholine (ACh) and cholecystokinin (CCK), depolarize the acinar cell membrane and I had provided some (indirect) evidence that this was associated with a rise in the cytosolic Ca2+ concentration ([Ca2+]i). In conjunction with a further paper3 from my sabbatical year at the University of Cambridge, in collaboration with John A Williams , this provided the earliest evidence indicating that exocrine pancreatic secretion is acutely controlled by changes in [Ca2+]i.It was a great honor for me to receive the Palade Prize and to deliver the Palade Prize Lecture on 7th July 2022, at the Joint meeting of the Japan Pancreas Society with the IAP, at Kyoto’s International Conference Center . I was p1 Secretin, the first hormone to be discovered,4 stimulates a bicarbonate-rich fluid secretion from the pancreatic duct cells. The first preparation of pure enzymes, including trypsin and chymotrypsin from the pancreas, was made by John Northrop, who received the Nobel Prize for this work.5Although much of Palade’s work was focused on the exocrine pancreas, it was highly relevant to cell biology in general. In fact, Palade was interested in understanding general cell properties, but had chosen the acinar cell of the pancreas as his study object because of its special characteristics. These included: the polarized structure of the acinar cells—enabling a clear spatial separation of the different elements of the secretory pathway, its large endoplasmic reticulum (ER) and the fact that the pancreatic acinar cell produces large quantities of protein. It was also important for Palade that previous work on the exocrine pancreas had led to work of general significance.2+. He felt that the well-established mechanism in nerve endings, namely that depolarization of the membrane opens Ca2+ channels,6 was likely to work everywhere, including in the exocrine glands. That a rise in [Ca2+]i is the principal activator of secretion, also in exocrine glands, is now well established, although the mechanism is different from what Palade thought, as discussed later in this article. However, there are exceptions. Palade had difficulties acknowledging the results of Michael Schramm and Zvi Selinger showing that in the parotid gland the main intracellular messenger activating exocytotic secretion is cyclic AMP.7 In this context, I witnessed clashes between Palade and Schramm at several conferences. Palade’s opinion that the depolarization-elicited Ca2+ influx is the activator of pancreatic secretion, undoubtedly influenced his collaborator George Scheele, who published experiments that were interpreted to confirm this view.9 This turned out to be wrong, as I shall discuss below in the section on physiological Ca2+ signals.Palade looked for mechanisms that were universally important and believed that nature uses such mechanisms in many different contexts. From my discussions with Palade, it was clear to me that he believed that exocytosis was universally activated by Ca11 My own primary interest was the acute regulation of secretion and to study this I needed methods for observing transport events in living cells with high spatial and time resolution. In this account, I shall present what I consider to be my key physiological and pathophysiological experiments on the exocrine pancreas, hoping that at least some of them will turn out to be of general relevance.Palade’s selection of the exocrine pancreas as his principal study object, was a lucky choice and paid off handsomely. He pioneered electron microscopical studies and enhanced them ingeniously with the so-called pulse-chase technique.11 However, with regard to acute dynamic events, the pancreas—like all exocrine glands—was a “black box.” It was my particular aim to understand the intracellular mechanism by which secretion is acutely switched on when acinar cells are stimulated by ACh or CCK. At that time (late 1960s), this was “terra incognita.”When I started research as an undergraduate medical student in Copenhagen in the late 1960s, histology and electron microscopy had already taught us much about the structure of the acinar cells. Furthermore, the pulse-chase experiments of George Palade and Jim Jamieson had, as already mentioned, revealed the crucial steps in the acinar secretory pathway.The acute activation of secretion was particularly striking in the case of my first study object, namely the cat submandibular gland. Cannulating the main duct from the gland and isolating the chorda tympani containing the parasympathetic nerve fibers to the gland, it was easy to observe the virtually instantaneous flow of saliva upon electrical nerve stimulation. Furthermore, the flow of saliva stopped immediately when the electrical stimulation was discontinued. I was intrigued by these acute events.12 which was seen as a useful general transport model. However, in the intestines, kidney, and frog skin, fluid and electrolyte transport occur continuously, in contrast to what happens in the exocrine glands. The only theory for control of exocrine secretion was based on acute Cl− pump regulation and the involvement of ion channels was explicitly rejected.In the 1960s, the field of epithelial transport physiology was dominated by work on the kidney and the intestines, essentially informed by the pioneering work of Hans Ussing on the frog skin,Physiological Reviews.15 Lundberg’s key experimental evidence, indicating that the ACh-elicited hyperpolarization of the acinar cell membrane was fundamentally different from inhibitory post-synaptic potentials in the nervous system, had been published in Nature a few years earlier.13 Lundberg found that the ACh-elicited membrane potential change did not exhibit a reversal potential and was not associated with any change in membrane conductance. Since it was dependent on the presence of Cl− in the external medium, he concluded that it must be due to direct activation of an electrogenic Cl− pump.14 Lundberg was a well-known neuroscientist, who had worked, and published many papers, with the Nobel Laureate John Eccles,17 so his work on the salivary glands was naturally regarded as state of the art and dominated the chapter on secretion mechanisms in Arnold Burgen’s 1961 Monograph for the Physiological Society on “Physiology of the salivary glands.”18 It is therefore understandable that when I and a fellow undergraduate medical student published a paper in 1968, in the relatively obscure journal Experientia,19 contradicting the work of Lundberg, it had virtually no impact. We showed that the ACh-evoked hyperpolarization was undiminished when Cl− in the perfusion fluid was replaced by sulphate,19 and we were right.Lundberg’s transport model for salivary secretion was published in 1958 as part of a review article in 2 did not, of course, agree with Lundberg’s model. Furthermore, after my return to Copenhagen from the sabbatical in Cambridge, I was able to show that the ACh-elicited depolarization of the pancreatic acinar cell membrane was associated with a marked reduction in membrane resistance  evoke depolarization, rather than hyperpolarization, of the pancreatic acinar cell membranesistance . It was 20 It was impossible, using the dissecting microscopes at our disposal, to see the cells into which the microelectrode had been inserted. However, all histological work on the exocrine pancreas indicated that the vast majority of cells were acinar, and we therefore assumed that our recordings reflected events in these cells. The other important assumption was that ACh and CCK only acted on acinar cells. In retrospect, these assumptions seem quite bold and are, in fact, not 100% correct but, in practice, it turned out that they were “good enough” and the results from this early period of glandular investigations (up to 1977) are reproducible. It was nevertheless always one of my goals that we should be able to see the cells we were investigating. Our work had actually little impact until we were able to do this.With regard to monitoring acute cellular events, an electrophysiological approach had always been attractive to me because of the high time resolution that can be achieved. However, it was a severe limitation that we originally had to work “blind” by simply inserting a sharp microelectrode into a glandular tissue and record changes in membrane potential.Direct visualization of the acinar cells in the living pancreas only became possible after I had moved to the University of Dundee in 1975. There were of course many phase-contrast microscopes available, with sufficient magnification to allow inspection of individual cells, but I needed a microscope with a long working distance so that there would be adequate access for the microelectrodes to be inserted into the tissue. With the help of the Chief Technician in the Physiology Department , I found the solution from a completely different field. A phase-contrast mirror objective with an impressively long working distance (2 cm), that had originally been designed for the inspection of metal surfaces, turned out to be perfect for my purpose. Using this microscope, my first PhD student, Noriyuki Iwatsuki  managed to record ACh-elicited membrane potential and resistance changes simultaneously from two neighboring acinar cells under direct visual control . The suc23 Interestingly, we also showed that fully coupled cells within the same acinus could be completely and reversibly uncoupled from each other with supramaximal ACh stimulation.23 Most importantly, we were able to show that intracellular injection of Ca2+ could mimic the effect of external ACh application.24 This was the earliest evidence for Ca2+-mediated opening of Cl− channels in the cell membrane. That these channels were selectively located in the apical cell membrane could only be proven many years later, when I was at the University of Liverpool. This, again, depended on direct visual inspection of the acinar cell under investigation. These difficult experiments25  of such channels per cell.29 These early papers, summarized in a review article in Nature published in 1984,30 proved in a way that was beyond dispute, that epithelial cells did have several types of ion channels with characteristics similar to those found in nerve and muscle cells. It is, however, important to stress that they do not possess voltage-activated Ca2+ channels.31 These studies also showed that the most important ion channels in exocrine gland cells were controlled by receptor-activated changes in [Ca2+]i.33 The work in this period resulted in the formulation of the first coherent model of acinar fluid secretion, focusing on Ca2+-activation of Cl− channels in the apical membrane and K+ channels in the basolateral membrane.34 I reviewed these data in detail, when I gave the Physiological Society’s Annual Review Prize Lecture in 1991, which was subsequently published in J Physiol.31The invention of the gigaseal high-resolution method for recording single channel and whole cell currents2+ for secretion, also in the exocrine glands, was already recognized in the early 1970s.36 Unfortunately, most studies of the exocrine glands were rather crude and had poor time resolution. As an electrophysiologist, I was always attracted to experiments in which events could be recorded continuously as they happened and during my period in Cambridge in the early 1970s, I was part of a team that developed an automated fluorescence method for measurement of amylase output from superfused small fragments of pancreas.37 With this technique, one can monitor continuously, and with relatively good time resolution, the enzyme secretion from the pancreas38 and observe directly the effect of, for example, stimulation with ACh  acting on the outside of a cell could cause opening of Ca2+ release channels in the ER had been solved in 1983 by the experiments of Irene Schulz and Michael Berridge on permeabilized pancreatic acinar cells, in which inositol 1,4,5-trisphosphate (IP3) had been shown to release Ca2+ from a nonmitochondrial store.39 IP3 very quickly became recognized as the ubiquitous intracellular messenger releasing Ca2+ from internal stores.40 Employing the Ca2+-activated Cl− channel in the apical cell membrane as an endogenous Ca2+ sensor .47 We therefore concluded that the guinea pig pancreatic acinar cell was electrically nonexcitable, exactly like the mouse pancreatic acinar cell, but possessed VIP receptors activating cyclic AMP production.47 More recently, we have been able to observe neuro-glandular transmission in the mouse pancreas, by measuring directly and simultaneously [Ca2+]i in a nerve ending and its adjacent acinar cell.48 K+ depolarization evoked a Ca2+ signal first in the nerve cell and then in the acinar cell. However, when atropine was applied, K+ depolarization only evoked a Ca2+ signal in the nerve cell.48It had become clear already in the early 1970s that the pancreatic acinar cells were electrically nonexcitable, because depolarizing the cell membranes by exposure of the pancreatic tissue to a solution with a high concentration of K2+] inside the ER ([Ca2+]ER), it could be seen that the predominant signaling pattern of repetitive short-lasting spikes could continue for a few minutes in the absence of external Ca2+, without any observable reduction of [Ca2+]ER. However, when [Ca2+]ER began to decline, the amplitude of the Ca2+ spikes decreased sharply and finally spiking ceased at a point when the ER was far from empty of Ca2+ , we concluded that the IP3 receptors on the ER must be concentrated in the apical region.49 When I presented these data at a seminar held at Tokyo University in March 1993, it transpired that Haruo Kasai from that institution had very similar data and had reached the same conclusion. Both of us had submitted papers reporting this finding to Science and both our papers had been rejected. At the time we met, we had both submitted our papers to Cell and were both battling with the editor to get them accepted. We were finally successful, and our papers were published in Cell, back to back, in August 1993.50Imaging revealed that the Cah region . Since, 2+ spikes were capable of triggering exocytotic enzyme secretion, we needed to employ a method for monitoring this process at high time resolution. This called for an electrophysiological approach. Neher and Marty had developed such a method, namely measurement of capacitance.53 The capacitance of cellular membranes is a biological constant and any insertion of membrane, as occurs in exocytosis, will increase the capacitance and the subsequent endocytic retrieval of the membrane will reduce the capacitance. By simultaneously recording membrane conductance and capacitance, it was possible to resolve the time course of the secretory process during an individual Ca2+ spike. As seen in 2+]i (monitored by the Cl− conductance) slightly preceded and slightly exceeded the capacitance rise, indicating that the Cl− channels are a bit more sensitive to a rise in [Ca2+]i than the exocytotic machinery. We also attempted to visualize the secretion process by monitoring the disappearance of the fluorescent agent quinacrine, which accumulates in acid compartments such as the ZGs. As seen in In order to test whether the short-lasting Ca2+ channels in the acinar cell membrane upon stimulation with either ACh or CCK.35 This explains the experimental observation that the initial part of the secretory response to stimulation is completely independent of the presence of Ca2+ in the external solution  current and has been studied extensively by Anant Parekh and others, particularly in immune cells.56 The CRAC channels are extremely Ca2+ selective and have a very low single channel conductance, making it impossible to record single channel currents.56 Even the maximally activated whole cell CRAC current, elicited by complete emptying of the ER Ca2+ store, is typically only a few pA 55 and therefore difficult to record in many cell types.There is no immediate opening of Casolution . However2+ pumps with thapsigargin, at the same time as he recorded the loss of Ca2+ from the ER  to look for mitochondria in living pancreatic acinar cells.62 Many EM pictures of acinar cells had been published in numerous articles that had not shown any particular distribution of the mitochondria, so it was a great surprise for us to see that in the living acinar cells there was a very distinct distribution of these organelles. By far the highest density of mitochondria was seen surrounding the apical granular region. The striking appearance of what we termed a mitochondrial belt .64 Having identified repetitive local Ca2+ spikes in the apical region as the physiological regulators of pancreatic enzyme secretion,51 later also shown in human acinar cells,65 the hypothesis that a sustained global [Ca2+]i elevation might be toxic seemed plausible.49Noting that a prolonged excessive rise in [Ca2+ signals to a global sustained [Ca2+] elevation, became the starting point for our subsequent focus on the role of mitochondrial inhibition in the development of AP. It would gradually become clear that excessive release of Ca2+ from internal stores, causing pathological opening of CRAC channels and therefore excessive Ca2+ entry, would overwhelm the mitochondria, and open a large channel in the inner mitochondrial membrane . The opening of this pore caused mitochondrial depolarization66 and thereby stopped ATP generation.67 This further exacerbated the toxic effect of the prolonged [Ca2+]i rise, by disabling the ATP-dependent Ca2+ pumps that normally extrude excess Ca2+ from the cytosol. It became abundantly clear that inhibition of mitochondrial function is a crucial step in the process of acinar necrosis that is such a critical element in the development of AP.66 It follows from these findings that preventing the opening of the MPTP should protect against the development of AP. This prediction has turned out to be correct. When MPTP opening was blocked pharmacologically or genetically, all the cellular changes associated with AP were abolished or severely reduced in four different models of experimental AP.68The experiment shown in 69 further developed many years later by Lampel and Kern,70 demonstrating vacuole formation inside acinar cells following hyperstimulation. Could this be related to toxic Ca2+ signals? We found that stimulation with nanomolar, rather than the physiological low picomolar, CCK concentrations, evoked a sustained global [Ca2+]i elevation and induced vacuole formation.71 Importantly, this vacuole formation could be prevented by loading the cells with a Ca2+ chelator or simply by omitting Ca2+ from the external solution.71 We also utilized a probe that becomes fluorescent after trypsin cleavage of two oligopeptide side chains and could in this way visualize, in the living tissue, the increase in intracellular trypsin concentration elicited by CCK hyperstimulation. Our live imaging of the subcellular distribution of trypsin activity showed that trypsin activity appeared initially in the apical granular area,71 as also reported at around the same time by Markus Lerch and his collaborators.72 However, the image resolution in both our study71 and the one from Lerch’s group72 was insufficient to determine the precise organellar localization.I was impressed by the early studies of Babkin,71 was not without problems, as is often the case, particularly with important new data. We first submitted the paper to Gastroenterology but, after several rounds of reviewing and revision, I had the feeling that we were not going to be able to persuade the journal to accept our study, so we decided to send the manuscript to PNAS. At that time, this was done via a member of the National Academy who, by mistake, used surface rather than air mail to send our paper from Cambridge in England to the Washington office of PNAS. It therefore took a couple of months before the paper arrived in the US. Happily, our bad luck then finally ended, and the paper was not only accepted, but also highlighted by an excellent commentary written by Anant Parekh.73The publication of our study74In order to progress, we formulated a very specific hypothesis, namely that the vacuoles were post-exocytotic endocytic structures and that the initial trypsinogen activation occurred in the resulting endosomes (vacuoles). To test this, it was essential to get the trypsin-sensitive probe into the endocytic compartment and this was accomplished by including the probe in the external bathing solution. Isolated acinar cells were then stimulated by a high (nanomolar) CCK concentration. As seen in 2+. The matrix may behave as an ion exchanger and if Ca2+ could be replaced by a mono-valent cation, it would disaggregate. This could happen after fusion of the ZG with the apical cell membrane and opening at the point of fusion, because the granule interior would then be in direct contact with the fluid in the acinar lumen, which has a high Na+ concentration.75 Ion exchange could be part of the normal physiological secretion process, as the pro-enzymes need to be free in order to become working enzymes in the gut. However, in the pathological state of hyperstimulation, the excessively high [Ca2+]i in the apical pole may provide an extra strong stimulus for endocytosis, so that the enzymes rather than being secreted, which would require fusion pore expansion,76 would be taken back into the cell. There is, to the best of my knowledge, no evidence for this hypothesis, but it is at least a plausible way of thinking about the process of intracellular trypsinogen activation.The biophysical mechanism underlying trypsinogen activation is still unclear. The major part of the content of the ZG is not in free solution but bound in the granular matrix, held together by Ca74 as well as those from the groups of Saluja77 and Lerch,72 was the exclusive use of CCK hyper-stimulation to induce AP-like changes in the acinar cells. AP in real life is not happening as a result of high (nanomolar) CCK concentrations in the blood, but mostly as a result of gallstone complications or alcohol abuse. CCK hyperstimulation could activate several G-proteins and may therefore result in intracellular actions that could be different from those elicited by the pathophysiologically more relevant actions of alcohol or bile acids. In later studies, we therefore discontinued the CCK hyperstimulation protocol and instead focused on ethanol.One valid criticism of both our studies,78 it turned out to be rather minor. Ethanol, even at the extremely high concentration of 850 mM, a much higher level than can be observed in blood after even the most extreme alcohol consumption,79 mostly only elicited a small elevation of [Ca2+]i. Remarkably, such a high ethanol concentration did not even prevent the acinar cells from regulating [Ca2+]i back to the resting level after a supramaximal ACh stimulus had caused a major rise. This showed that the ATP-dependent Ca2+ pumps were fully functional in the presence of a very high alcohol concentration.78I had anticipated that exposure of isolated pancreatic acinar cells to high concentrations of ethanol would be destructive but, when we first tested the effect of ethanol in 2003,Science in 1988 by Laposata and Lange,80 indicating that the toxic effects of ethanol were mainly due to the action of nonoxidative ethanol metabolites rather than ethanol itself or its oxidative metabolite acetaldehyde. A simple hypothesis now suggested itself, namely that the prolonged and excessive [Ca2+]i elevations that were destroying the pancreatic cells in AP were, in the case of alcohol-related AP, caused by the product of ethanol and fatty acids. Fatty acids can combine with ethanol to yield fatty acid ethyl esters (FAEEs) and this process is particularly effective in the exocrine pancreatic cells.80 We thus began to test the actions of palmitoleic acid (POA) and palmitoleic acid ethyl ester (POAEE) and quickly established that POAEE was a powerful releaser of Ca2+ from internal stores, eliciting a substantial and sustained elevation of [Ca2+]i.78 The mechanism by which POAEE releases Ca2+ from the ER, and also from acid stores81 is still unknown, but depends on functional IP3 receptors, as shown by knock-out experiments.81 The action of POAEE could be mimicked by many other FAEEs78 but, for practical reasons, we concentrated our efforts on studying the effects of POAEE. In contrast, acetaldehyde did not cause any Ca2+ signals and did not destroy the acinar cells.78 We have not studied the effects of bile acids as intensively as those of alcohol and fatty acids, but they are rather similar to what has been observed with POAEE.83We then found an important paper published in 2+]i plateau depended on Ca2+ entry through CRAC channels and pharmacological inhibition of the opening of these channels markedly reduced the POAEE-elicited [Ca2+]i rise .99 These names are clearly related to those of astrocytes (Astron [Greek] meaning star).100 Astrocytes, hepatic and pancreatic stellate cells have in common that they were originally regarded as relatively passive in their respective organs. The situation for the astrocytes, in particular, has changed dramatically in recent years and there is now a huge literature on their many and highly important functions.100It is of course obvious that immune cell function must be critical to the development of the inflammatory response in AP, but until very recently there had been no attempt to visualize directly activity in non-acinar cells in the acinar environment. Morphological studies of the exocrine pancreas have overwhelmingly been focused on the acinar cells. The stellate cells, which are the closest neighbors to the acinar cells, represent a very small volume of the pancreas, can hardly be seen with standard light microscopy and rarely make an appearance in EM pictures. When the pancreas is digested by collagenase, as is routinely done when studying isolated acinar cells or cell clusters, one essentially only sees acinar cells. However, when watching living cells in quasi-intact small pieces of pancreas, and especially with fluorescence microscopy, one can observe that small and thin, but relatively long, cells are located on the basal acinar surface . These a2+ signals in response to stimulation with bradykinin (BK), but not with ACh  101 than those required in the pancreatic stellate cells  inhibited the development of the cellular changes that are characteristic for AP.103BK is a small inflammatory peptide that is split off from the large bradykininogen molecule by the action of the protease kallikrein, which is produced in the acinar cells. Many years ago, Ryan, Moffat and Thompson from Montreal General Hospital proposed that BK could play an important role in the development of AP.2+ signals in the stellate cells  is sufficient to evoke a clear Ca2+ signal and a near-maximal response occurs at BK concentrations that have been measured during AP  receptor antagonists markedly reduce the extent of necrosis evoked by FAEEs and bile acids.97 Furthermore, a couple of days after induction of alcohol-related experimental AP, it was apparent that there had been a desensitization of the response of the stellate cells to BK .104 It would appear that this has a deleterious effect on the neighboring acinar cells, because pharmacological inhibition of the Ca2+-sensitive NO synthase markedly reduced the extent of necrosis evoked by bile.104 The mechanism by which NO acts, directly or indirectly, is currently unknown. Overall, the live imaging work on stellate cells in the quasi-intact pancreas has provided compelling evidence for an active role of these cells in early stages of AP, although many mechanistic aspects remain unclear.Live imaging showed that Ca2+ signals in the stellate cells occur by a mechanism that is very similar to the one described for the action of ACh on the acinar cells. The initial steep rise in ([Ca2+]i) is independent of the presence of external Ca2+ and therefore due to release from internal stores. However, unlike the action of ACh on the acinar cells, this initial rapid spike-like [Ca2+]i rise is always followed by a prolonged elevated [Ca2+]i plateau  as well as MRS 2365 (a highly selective P2Y1 agonist) mimicked the effects of ATP and ADP , IgG elicited substantial repetitive Ca2+ spikes, which were longer than the occasional single spike that could occur in macrophages in the normal healthy pancreas.105The number of macrophages in the pancreatic tissue increased markedly after induction of alcohol-related AP in mice. Three days after start of injection of ethanol and POA, the density of macrophages was six times higher than in the normal pancreas, whereas the density of stellate cells was unchanged.2+ signals elicited in the pancreatic macrophages by ADP or ATP were due to primary release of Ca2+ from intracellular pools followed by Ca2+ entry through CRAC channels.105 The Ca2+ signals in the macrophages were, however, more quickly reduced in the absence of external Ca2+ or in the presence of CRAC channel inhibition than, for example, the ACh-elicited Ca2+ signals in the acinar cells.105 The acinar cells do of course have a very large ER, and therefore a much larger intracellular Ca2+ store than most other cell types.The Ca2+ signal generation in the endogenous macrophages in the pancreas. However, based on evidence from immune cells isolated from the blood,107 it seems highly likely that the Ca2+ signals in the pancreatic macrophages will stimulate secretion of inflammatory cytokines.So far, we have no direct evidence concerning the functional consequences of the Ca2+ signals in response to BK stimulation (2+ signals in the stellate cells.105 Because BK levels in plasma are elevated in AP, some of the macrophages that invade the pancreas in the initial phase of the disease may also, as is the case for the stellate cells, be further stimulated by BK and in this way participate in both the cytokine and BK storms that are characteristic of severe AP.105 From a therapeutic perspective, it is important that all Ca2+ signals in pancreatic macrophages can be inhibited by pharmacological CRAC channel inhibitors, as is also the case for the acinar and stellate cells.105 The remarkable effectiveness of CRAC channel inhibition in protecting against experimental AP is therefore almost certainly due to actions on all three cell types.87Although the majority of the pancreatic macrophages did not display Camulation , there w109 The virus has been shown to enter many different cell types in the body. These include cells in the respiratory tract, the nervous system and the gastrointestinal tract.87 The cytokine and BK storms cause massive inflammation, including severe vasodilatation and extravasation, impeding oxygen transfer from the alveoli to the blood. We were therefore interested in testing whether SARS-CoV-2 might act directly on macrophages and/or stellate cells in the pancreatic tissue. As seen in 2+ spikes in both endogenous macrophages and stellate cells, whereas no effects were observed in the acinar cells.113 All Ca2+ signals could be blocked by CRAC channel inhibition.113SARS-CoV-2 entry into cells occurs via binding of its S protein subunit to ACE2 receptors allowing endocytic virus uptake.2+ signal generation in the macrophages was longer than in the stellate cells (2+ signals in the macrophages were generated secondarily to events in the stellate cells. It turned out that an interleukin binding protein could prevent the SARS-CoV-2 spike protein from generating Ca2+ spikes in the macrophages, but not in the stellate cells (It was noticeable that the latency for Cate cells . This mite cells .2+ signals in the acinar cells already described (2+ signals in the stellate cells.114 Thus, toxic Ca2+ signals in the stellate cells may not necessarily be secondary to toxic Ca2+ signals generated in the acinar cells, but can also occur as a primary event. The stellate cells may therefore play an even bigger role in the development of AP than hitherto thought87. Furthermore, it has now been shown that the pressure-sensitive Piezo-1 channels are functional also in stellate cells.115 The mechanism, already described in acinar cells, whereby physical pressure can elicit small Ca2+ signals mediated via Piezo-1 channels that subsequently lead to opening of TRPV4 channels, generating a more substantial Ca2+ influx, also appears to operate in the stellate cells.115 Physical pressure could therefore evoke Ca2+ signals in both acinar and stellate cells.More work clearly needs to be carried out on the mechanism of action of SARS-CoV-2 on pancreatic stellate cells, but at least the experiments conducted so far  highlighescribed , is capa117 we can expect much enlightenment in the coming years.Acute observations in real time of signaling and secretion events in identified cell types in the exocrine pancreas have provided valuable information about physiological and pathophysiological processes in this gland. The mechanisms underlying the principal functions of the acinar cells have been particularly well investigated and are now reasonably well understood, whereas studies of the stellate and immune cells are still in their infancy. Future studies of pancreatic pathophysiology will almost certainly depend on direct imaging studies of these cells and their interactions with each other in the intact pancreas in situ. With the recent spectacular developments in in vivo imaging technology,


ADGRD1 (GPR133), an adhesion G protein-coupled receptor (GPCR), has been linked to cancer. However, the prognostic value and regulatory function within non-small-cell lung cancer (NSCLC) is still unclear. This work adopted various bioinformatics methods, including publicly available databases as well as real-time PCR (RT-PCR), for detecting ADGRD1 expression level and investigating the correlation between ADGRD1 expression level and prognosis, tumor mutational burden (TMB), microsatellite instability (MSI), immune infiltrating cells, immune-related genes, and targeted regulation mechanisms in NSCLC. According to the results, ADGRD1 expression decreased within NSCLC, which might be the factor predicting prognosis of NSCLC. Meanwhile, ADGRD1 showed significant correlation with TMB and MSI, respectively, as well as immune cell infiltrating levels in lung adenocarcinoma (LUAD), which were primarily linked to macrophage M1, mast cell resting, T cell CD4 memory activated, and T cell CD4 memory resting and were associated with mast cell activated and mast cell resting in lung squamous cell carcinoma (LUSC). The most promising upstream regulation pathways of ADGRD1 were likely miR-142-5p, miR-93-5p, and miR-17-5p, which were overexpressed and associated with poor prognosis in NSCLC. ADGRD1 and immune-related genes correlated with ADGRD1 were shown to be enriched in “positive regulation of leukocyte activation,” “external side of plasma membrane,” “receptor ligand activity,” and “cytokine-cytokine receptor interaction” pathways. ADGRD1 expression and regulation may be critical in determining NSCLC prognosis. Cancer represents a major factor causing global mortality, endangering human health and life safety. Lung and bronchus cancer accounts for 235,760 newly diagnosed and 131880 death cases in the United States by 2021, making it the main cause of death in patients and the survival rate at 5 years is just about 21% [The GPCR adhesion family is extensively expressed in human tissues and important in human physiological processes. GPCRs are linked to several diseases –5, implyIn a nutshell, TMB indicates the overall mutation number within tumors , 9. TMB Previously, biological function of ADGRD1 in different human cancers has not been completely explored and only a few researches on ADGRD1 analysis in NSCLC have been published. Multiple databases were used in this study for exploring the prognostic significance of ADGRD1 within pan-cancer. In addition, ADGRD1 expression and the link between TMB, MSI, and immune infiltrating cells were investigated. Furthermore, the relationship between ADGRD1 and immune genes was investigated, signaling pathways with relevant immune genes. Finally, the microRNAs controlled by ADGRD1 were predicted, and the relationship between these miRNAs and NSCLC prognosis was examined.http://xena.ucsc.edu/), a convenient database for obtaining TCGA database data. The inclusion criteria were as follows: completed ADGRD1 mRNA sequencing data, completed somatic mutations data, and detailed clinical follow-up information. R packages “plyr” and “ggpubr” were applied to draw the boxplot of gene expression sequences. Pan-cancer ADGRD1 expression analysis was also performed by GEPIA2 (http://gepia2.cancer-pku.cn/) database which contained TCGA and GTEx data.RNA sequences, somatic mutations, and survival data associated with a total of 33 cancers were downloaded from UCSC Xena , disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI). The Cox regression analysis evaluated the association between survival time and survival status by considering gene expression as a continuous variable. Concurrently, hazard ratio (HR) with the 95% confidence interval was considered. An HR value greater than one indicated that ADGRD1 was a risk factor rather than a protective factor in this cancer. The Kaplan-Meier approach was utilized to classify cases as the high-expression or low-expression group according to their median level. They analyzed the different OS of both the expression groups. The R packages “survival” and “survminer” were used to draw the Kaplan-Meier curves.TMB is the overall mutation number within DNA carried via cancer cells. MSI indicates contaminant nucleotide gain/loss in repetitive DNA fragments. MSI score could be analyzed based on the data of TCGA. The radar map containing the correlation between ADGRD1 expression level and TMB or MSI was performed by “fmsb” R package.This work adopted ESTIMATE algorithm to determine immune, stromal, and ESTIMATE scores with “limma” and “estimate” packages in R software, for predicting the purity of different cell types within the tumor microenvironment. The estimation of immune infiltrating cells in NSCLC was analyzed using CIBERSORT . Coexprehttps://www.immport.org/home) for further screening genes significantly coexpressed with ADGRD1 in both LUAD and LUSC (P < 0.001 and |R| > 0.2 as the cut-off criterion). After screening, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed by ADGRD1 and screened genes. R packages “org.Hs.eg.db,” “clusterProfiler,” “enrichplot,” and “ggplot2” were utilized to show the result of GO and KEGG analyses with the cut-off threshold P value = 0.05 and q value = 1.We downloaded 1793 immune-related genes from ImmPort database (https://starbase.sysu.edu.cn/) was used. MicroRNA expression and clinical information from NSCLC cases were downloaded in TCGA . The inclusion criteria were as follows: (1) completed microRNA sequencing data and detailed clinical follow-up information; (2) miRNAs from starBase database with the screening threshold as programNum > = 2 and targeted ADGRD1. The exclusion criteria were as follows: miRNAs not negatively correlated with ADGRD1. The “survival” package of R language was used in identifying the prognostic microRNAs. And the cut-off criteria of differential expression were |log2fold change| = 1 and diffPval = 0.05.To predict upstream microRNAs that could regulate ADGRD1 expression, starBase database , whereas NCI-H1299 human NSCLC cells within RPMI-1640 medium that contained 10% FBS. Following Takara RR820A kit instruction, RT-PCR was carried out, with U6 being the endogenous control. Takara Biomedical Technology  designed and synthesized ADGRD1 primer. Method of 2https://www.proteinatlas.org/) covers protein levels in tissues, cancers, and normal cells. IHC was applied to validate ADGRD1 expression levels within normal lung and lung tumor tissues.The HPA project . All correlation analyses were carried out by the Spearman method .The Wilcoxon test was applied to calculate the difference between normal tissues and tumor tissues about ADGRD1 expression with the cut-off standard of First, we analyzed the expression of gene ADGRD1 in different cancers and found that gene ADGRD1 was differentially low-expressed in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), LUAD, LUSC, prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC) between normal tissue and tumor tissue data from TCGA . MeanwhiP = 0.002; DSS: HR = 0.777, 95CI% from 0.650 to 0.928, P = 0.005; DFI: HR = 0.743, 95% CI from 0.599 to 0.922, P = 0.007; PFI: HR = 0.846, 95% CI from 0.741 to 0.966, P = 0.014), while a risk factor in LUSC . Kaplan-Meier survival curves also showed that ADGRD1 expression was correlated with the prognosis in LUAD  and LUSC ; meanwhile, the significant results were displayed in First, we used the Cox regression to analyze the impact of ADGRD1 expression among OS, DSS, DFI, and PFI in pan-cancer . The resP = 0.019), LGG (P = 0.001) and thymoma , while negatively related to TMB in BLAC (P = 0.048), BRCA (P < 0.001), COAD (P = 0.019), KIRC (P = 0.025), KIRP (P = 0.016), LIHC (P = 0.029), LUAD (P < 0.001), LUSC (P = 0.012), pancreatic adenocarcinoma , PRAD (P = 0.007), SKCM (P = 0.010), STAD (P < 0.001), and UCEC (P < 0.001). Furthermore, we calculated the correlation between ADGRD1 expression and MSI across cancers (P = 0.001), KIRP (P = 0.022), LUSC (P = 0.020), SARC (P = 0.042), STAD (P < 0.001), and UCEC (P < 0.001). According to the results, the expression of ADGRD1 was both correlated with TMB and MSI in LUSC, however, only significantly correlated with TMB in LUAD.TMB and MSI have been regarded as meaningful predictors of immune response. In the analysis of the association between ADGRD1 expression and TMB, MSI in pan-cancer is essential. As shown in  cancers . ADGRD1 P = 0.567; ImmuneScore, P = 0.007; ESTIMATEScore, P = 0.190). Varieties of immune infiltrating cells were contained in the tumor microenvironment. Thus, we calculated the association between ADGRD1 expression and immune cell infiltration levels in NSCLC. In LUAD, ADGRD1 was mainly related to 4 immune cells  in LUAD and associated with 2 immune cells (mast cell activated and mast cell resting) in LUSC (Figures We explore the association between tumor immune microenvironment, immune infiltrating cells, and ADGRD1 expression. The ESTIMATE algorithm including the StromalScore, ImmuneScore, and ESTIMATEScore was utilized to evaluate the association between ADGRD1 expression and different types of infiltrating cells in the tumor immune microenvironment across cancers and the results were shown in Table  Figures . The sigWe used ImmPort database to obtain the immunologically relevant list with a total of 1793 genes. After analysis, only 149 immune-related genes had significant differences correlated with ADGRD1 in NSCLC . ThroughWe used starBase database to predict the miRNAs targeting ADGRD1, and 20 upstream miRNAs were discovered. The inclusion criteria of miRNAs were significantly correlated with ADGRD1, differentially expressed, and the prognosis was statistically significant in LUAD or LUSC. After a series of screening criteria, as shown in Compared with BEAS-2B cells, the expression level of ADGRD1 mRNA was lower in NCI-H1299 cells, which is consistent with the prior research .As shown in Pan-cancer analysis is of great significance for comparing the role of the same gene in different cancers and provides a new direction for new tumor biomarkers, understanding the mechanism of tumorigenesis and development from the prevention and treatment of tumors. The same gene has the same or different effects on different tumorigenesis, mutations, tumor microenvironment, and copy number changes, which can be vital and helpful to diagnose and treat different tumors. CD161 pan-cancer analysis suggests that CD161 is the possible antitumor biomarker to develop the novel agents . In addiAccording to this research, ADGRD1 expression in pan-cancer was investigated using UCSC Xena database and GEPIA, and it was discovered that ADGRD1 expression level was relatively high in NSCLC cases compared to other cancers, particularly in NSCLC tissues, where ADGRD1 expression remarkably decreased compared with normal tissues. RT-PCR revealed that, compared to normal lung epithelial cells, ADGRD1 expression declined within NSCLC cells; meanwhile, immunohistochemical results of ADGRD1 obtained from the HPA revealed that compared to normal tissues, ADGRD1 protein expression decreased within NSCLC tissues, conforming to prior findings. According to our pan-cancer analysis, ADGRD1 had a critical effect on prognosis of different cancers. Few studies have reported the role of ADGRD1 in NSCLC prognosis. In this study, ADGRD1 downexpression was linked to a worse prognosis in LUAD and a better prognosis in LUSC, implying that ADGRD1 could be a viable prognostic biomarker in NSCLC.TMB has been demonstrated to be an independent predictor of immunotherapy response in various cancers, as well as a predictor of patient clinical outcomes , 19. MSIThe tumor microenvironment has become a popular issue in tumor research. Immune cells have critical effects on tumor microenvironment . Plasma Abnormal immune-related gene expression is strongly associated with immune infiltrating cells in various ways, making it a potential diagnostic and therapeutic target that provides new avenues for studying tumor molecular mechanisms of tumors –32. AddiBy understanding tumor-related biological pathways, microRNAs have provided critical hints for developing active diagnostic and therapeutic targets or unique prognostic biomarkers. The miR-6845-5p/miR-4455-ADGRD1 pathway was related to gastric cancer incidence . miR-142However, this study has several drawbacks. First, bioinformatics analysis of ADGRD1 was performed using several databases, which may introduce systematic bias. Second, although RT-PCR technique was involved for verifying ADGRD1 expression within NSCLC, it would be more precise to carry out in vitro/in vivo experiments and even clinical research to prove the prognostic role of ADGRD1in this study. Third, despite the expression of ADGRD1 was detected to be associated with tumor-infiltrating immune cells and prognosis of patients, we were unable to confirm the exact mechanism of the prognostic features. Therefore, future prospective studies focusing on ADGRD1 expression and immune infiltrating cells may be needed to solve this issue.In summary, we found that ADGRD1 showed relationship to the clinical outcomes, TMB, MSI, and immune infiltrating cells in multiple cancers. In NSCLC, ADGRD1 expression was lowered and significantly associated with prognosis, TMB, MSI, tumor microenvironment, and immune infiltrating cells. Additionally, the upstream miRNAs of ADGRD1, ADGRD1 associated immune-related genes, together with ADGRD1 and its associated immune-related genes enriched pathways were defined in NSCLC. As a result, the expression and the mechanism regulation of ADGRD1 might be the prognostic markers in NSCLC.


Neoantigens derived from non-synonymous somatic mutations are restricted to malignant cells and are thus considered ideal targets for T cell receptor (TCR)-based immunotherapy. Adoptive transfer of T cells bearing neoantigen-specific TCRs exhibits the ability to preferentially target tumor cells while remaining harmless to normal cells. High-avidity TCRs specific for neoantigens expressed on AML cells have been identified in vitro and verified using xenograft mouse models. Preclinical studies of these neoantigen-specific TCR-T cells are underway and offer great promise as safe and effective therapies. Additionally, TCR-based immunotherapies targeting tumor-associated antigens are used in early-phase clinical trials for the treatment of AML and show encouraging anti-leukemic effects. These clinical experiences support the application of TCR-T cells that are specifically designed to recognize neoantigens. In this review, we will provide a detailed profile of verified neoantigens in AML, describe the strategies to identify neoantigen-specific TCRs, and discuss the potential of neoantigen-specific T-cell-based immunotherapy in AML. Acute myeloid leukemia (AML) is the most common indication for hematopoietic stem cell transplantation (HSCT) , 2. The Recent developments in sequencing technology have revealed that a stepwise accumulation of genetic abnormalities is a hallmark of cancer. Gene fusions, small insertions and deletions (indels), non-synonymous point mutations, and cancer-specific splicing variants can result in the production of neoantigens Table  16]. On. On16]. In general, the identification of neoantigens comprises the following procedures. The first step is usually to compile a list of genomic alterations  from next-generation sequencing data, including whole-genome sequencing, whole-exon sequencing, RNA-sequencing, and DNA panel data, and convert them into “neopeptides” with appropriate lengths. The second step is to predict the binding affinity between neopeptides and patient-specific HLA alleles. Many tools are available for this prediction, such as BIMAS  for HLA Here we summarize hotspot neoantigens in AML.Gene fusions caused by chromosomal translocations play a significant role in AML, and they are implicated in approximately half of AML patients. Most gene fusions contribute to oncogenicity by altering hematopoietic-specific transcription factors, thus blocking myeloid differentiation at a specific stage. Due to the recurrent nature of fusion genes in AML, neoepitopes derived from these fusion genes may serve as efficient neoantigens for immunotherapy in AML patients.*40:01 donors. CD8+ T cell clones induced by this neopeptide kill AML cell lines and primary human AML cells in vitro and in an in vivo patient-derived murine xenograft (PDX) model, in a CBFβ-SMMHC-specific and HLA-A*40:01-restricted manner . T. T127]. 0052520) . In a suortality . In anot2 months , 131. Any switch . These eG12D [Despite the lack of clinical studies for neoantigen-specific TCR-T cell-based therapy in AML, deep and durable tumor shrinkage has been observed in a heavily pretreated patient with metastatic pancreatic ductal adenocarcinoma who received an infusion of autologous T cells transduced with a TCR specific for mutant KRASG12D . This fiAnother engineered T-cell-based therapy known as CAR-T is frequently employed in clinical trials to treat relapsed or refractory AML patients . Both TCThe TCR complex is a heterodimer consisting of alpha and beta chains. Unlike TCR, CARs are synthetic receptors created from a combination of an antibody-derived single-chain variable fragment (scFV), a T-cell receptor-derived CD3ζ domain, a transmembrane domain, and one or more intracellular co-stimulatory domains  that have evolved across five generations . Other tThe scFV domain of CAR-T cells binds to antigens expressed on the surface of cancer cells in an MHC-independent manner, therefore, targets for CAR-T therapy are all surface proteins, such as CD33 , CD123 , 140, NKModified T cells are potential for “On-target, off-tumor” toxicity in cases where the antigenic target is co-expressed on normal cells, such as CAR-T cells targeting leukemia-associated antigens and TCR-T cells targeting TAAs . NeoantiAlthough neoantigen-specific T cell-based immunotherapy has shown promising potential in treating AML patients without harming normal tissues, there are still challenges ahead for the application of TCR-T cells. First, the accurate identification of neoantigens presented by HLA molecules on the cell surface is a major obstacle. LC coupled to MS/MS has proved to be a fast and less biased technology to advance the identification of endogenously processed and presented HLA-ligands, although sample handling methods, instrumentations, and data searching algorithms are still required to be improved. Currently, integrating MS HLA-ligand profiling with other “-omic” technologies, such as whole exon sequencing and RNA-sequencing, is indeed of help to identify tumor-specific antigens for AML immunotherapy . Second,TCR-T cell-based immunotherapy for hematologic malignancies has made significant strides, especially in the treatment of high-risk AML. The major challenge is identifying appropriate targets. A variety of potential TAAs are being studied in preclinical or clinical settings as targets for TCR-T cell therapy, but special attention must be paid to the adverse effects caused by “off-targets”, because TAAs are also expressed in a small number of normal tissues. Neoantigens seem to be the best candidates for TCR-T targets in this regard since their expression is limited to AML cells. Encouragingly, high-avidity TCRs specific for mutant NPM1 and CBFB-MYH11 have been identified from the repertoires of healthy donors. T cells that have been genetically modified to express these TCRs shown specific killing abilities against AML cell lines as well as blast cells harboring mutated NPM1 and CBFB-MYH11, respectively. Given that the majority of AML patients exhibit these neoantigens, the results of future clinical trials are expected. Further research is needed into TCR-T cells that are directed against additional recurrent somatic mutations in AML, including IDH1/IDH2, FLT3-ITD, DNMT3A, and AML1-ETO.In conclusion, neoantigen-specific TCR-T cell therapy has good potential to treat AML. Illustrating the interaction between TCRs and neoantigens that are responsible for T cell reactivity, anti-leukemic responses, and clinical effects is the cornerstone. The security and efficacy of genetically modified TCR-T cells are vital as well. The present article reviewed advances in neoantigens for AML and proximal new techniques which can identify neoantigen-specific TCRs and activate a T-cell response. There are several restrictions, nevertheless, that need to be addressed in further study.


The NCX gene orthologs, isoforms, and their splice variants are expressed in a tissue-specific manner and exhibit nearly 104-fold differences in the transport rates and regulatory specificities to match the cell-specific requirements. Selective pharmacological targeting of NCX variants could benefit many clinical applications, although this intervention remains challenging, mainly because a full-size structure of eukaryotic NCX is unavailable. The crystal structure of the archaeal NCX_Mj, in conjunction with biophysical, computational, and functional analyses, provided a breakthrough in resolving the ion transport mechanisms. However, NCX_Mj  cannot serve as a structure-dynamic model for imitating high transport rates and regulatory modules possessed by eukaryotic NCXs. The crystal structures of isolated regulatory domains (obtained from eukaryotic NCXs) and their biophysical analyses by SAXS, NMR, FRET, and HDX-MS approaches revealed structure-based variances of regulatory modules. Despite these achievements, it remains unclear how multi-domain interactions can decode and integrate diverse allosteric signals, thereby yielding distinct regulatory outcomes in a given ortholog/isoform/splice variant. This article summarizes the relevant issues from the perspective of future developments.The plasma-membrane homeostasis Na The directionality of net Ca2+ fluxes depends on the membrane potential (Em) and ENCX (including ion concentrations and stoichiometry of ion exchange) i, [Ca2+]i, and membrane potential during the action potential influence the directionality of the net Ca2+ movements through NCX, since Em alternates between −90 mV and +50 mV) [+]i foster the reverse mode of NCX, whereas hyperpolarization or elevated [Ca2+]i favor the forward mode of NCX. Thus, NCXs’ directionality and transport rates are controlled by transmembrane gradients of Na+ and Ca2+, ion-exchange stoichiometry, and membrane potential. The effects of these factors become very dynamic during the action potential, where the directionality of charge/ion fluxes reverses and ion-exchange rates permute (up to 50-fold) within a few milliseconds i can dramatically affect ENCX, since any changes in [Na]i are powered in the third degree due to the 3Na+:1Ca2+ stoichiometry  through STIM1 and TRPC6 coupling [2+-influx usually occurs when a rapid Na+-influx (through a given channel or transporter) elevates [Na+]i within a restricted space i can raise the mammalian NCX currents ~25-fold, whereas the removal of cytosolic Ca2+ results in a slow inactivation termed I2-inactivation or Ca2+-dependent inactivation [2+-dependent increase in peak current occurs at lower [Ca2+] levels (~0.2 μM) -dependent activation curve, so much higher concentrations of Ca2+ are required for NCX1.1 activation  undergoes relatively small changes [2+ binding (Kd ≈ 0.2–0.5 µM) [2+-dependent activation of mammalian NCXs [2+ binding to CBD1), the Ca2+ binding to CBD1 of full-size CALX1.1 results in inhibition [Structural studies and Ca at CBD2 ,123,124.ectively ,41,42. T changes ,119,120.–0.5 µM) , thus reian NCXs ,119,120.ian NCXs ,42. The hibition ,172,173.d values of Ca2+ binding to the high-affinity sites of CBD12 are comparable among the cardiac (ACDEF), brain (AD), and kidney (BD) splice variants of NCX1, the Ca2+ dissociation rates from the high-affinity sites differ up to 200-fold [−1) for the occluded Ca2+ dissociation from the high-affinity regulatory sites of CBD12 obtained from the different isoform/splice variants of NCX and CALX. These slow Ca2+ off-rates correlate remarkably with slow inactivation kinetics of full-size NCX variants, which were measured using patch-clamp techniques upon removal of the cytosolic Ca2+ [2+ can be observed only in the CBD12 constructs (the two-domain tandem) but not in the isolated CBD1 or CBD2 ones—this means that specific synergistic interactions between the two regulatory domains generate a slow dissociation of occluded Ca2+ [2+ occlusion at the high-affinity C3 and C4 sites of CBD1 results in the Ca2+-dependent tethering of CBDs through a hydrogen-bonding network [2+ (due to the Ca2+ driven tethering of CBDs) can couple diverse regulatory phenotypes in NCX and CALX.Although the K200-fold ,123,124.lic Ca2+ ,169,171.ded Ca2+ ,123,124. network ,123,124.2+ binding sites of CBD2 are ~5.5 Å apart, where K585 (a homolog to E454 in CBD1) forms a salt bridge with D552 and E648 (in the absence of Ca2+), thereby yielding a relatively more stable apo-CBD2 structure [2+ binding sites at CBD2 [2+ binding affinities at both the CBD1 and CBD2 domains and modulates the Ca2+ dissociation rates from high-affinity regulatory sites to control NCX responses to cell-specific Ca2+ signaling/homeostasis [2+ binding to CBD2 . These structure-controlled arrangements significantly impact the regulatory capacity since the B-exon-dependent prevention of Ca2+ binding to CBD2 aborts the Ca2+-dependent alleviation of Na+-induced inactivation (2+ affinity at the CaI site (Kd ≈ 100 µM) [+-induced inactivation of NCX2, the effect of potential Ca2+ binding to CBD2 cannot be tested. In NCX3, B-exon replaces K585 with Glu to generate three Ca2+ binding sites at CBD2, whereas K585 in the A-exon of NCX3 prevents Ca2+ binding to CBD2   B.2+.Like mammalian NCXs, CALX1 also undergoes alternative splicing at CBD2, although the splicing segments of CALX1.1 and CALX1.2 are much shorter and differ from each other by only five residues ,65. Stru2+-dependent tethering of the CBD1 and CBD2 domains [2+ off-rates and decreases the affinity of Ca2+ binding (up to 10-fold) at the high-affinity Ca3-Ca4 sites of CBD12, either in NCX or CALX [2+ and alters the interdomain movements of CBDs [2+-dependent regulation of NCX ion currents [2+-dependent interdomain tethering/coupling of CBDs in NCX and CALX.Comprehensive analysis of mutants using stopped-flow assays revealed that a short interdomain linker (501-HAGIFT-506) connecting the two CBDs is essential for structure-based regulatory coupling ,116,117. domains ,42. Gene or CALX ,123,124. of CBDs ,42  between the CBD1 and CBD2. Notably, the Ca2+ coordination chemistry is very similar in isolated CBD1, CBD2, and CBD12 domains, derived from either NCX or CALX orthologs. However, a few structural differences have a primary mechanistic significance [2+ ligation at the Ca2, Ca3, and Ca4 sites in the CBD12 of NCX1-AD, CALX1.1, or CALX1.2. The most important structural difference between the isolated CBD1 and CBD12 is that D499 forms bidentate coordination with Ca4 in CBD12 in contrast with monodentate coordination in isolated CBD1. The significance of these structural arrangements is that they may predefine the structural stability of Ca2+-dependent tethering of CBDs in NCX and CALX [The discovery of the two-domain tandem (CBD12) crystal structures ,42 proviificance ,42, as sct area ~60 Å2 betand CALX ,123,124.2+ [2+-related tethering (through D499 and D500) contributes to two Ca2+ coordination at Ca3-Ca4 while concomitantly stabilizing the CBD interface  of the interdomain realm can be gradually modulated by varying composition of exons [In general, the crystal structures of NCX1-CBD12 and CALX1-CBD12 revealed three regions , which involve over 20 residues ,172,173.2+ . Notablynterface . Thus, tvariants ,123,124.of exons ,123,124.2+-binding EF loop of CBD1, the interdomain linker, and the FG loop of CBD2. The crucial point is that F450 interacts with H501, I628, A629, M631, and G632  to form a tightly packed hydrophobic core [2+-dependent interaction of F450 with H501 may limit the flexibility of the interdomain linker. Moreover, neighboring interactions between the CD and EF loops of CBD1 and the FG-loop of CBD2 (including the splicing segment) can modulate the Ca2+ binding affinity to the Ca3-Ca4 sites of CBD1. Since the FG-loop of CBD2 is unstructured (except for a short α-helix region at the C-terminus of the FG-loop), it is reasonable to assume that the side chains of a canonical α-helix impact the structure-dynamic features of the two-domain interface [The hydrophobic region at the two-domain interface contains residues that locate on the Cabic core . Notablynterface . Notablynterface . This stnterface   of CALX can more effectively stabilize the interdomain linker and CBD2 folding as compared with mammalian NCX variants, having a straight and longer α-helix at the matching position. These “minute” differences between NCX1 and CALX1 in the structural organization of the FG-loop α-helix can differently affect the rigidity of the interdomain linker and CBD2 folding [The α-helix region (belonging to the FG-loop of CBD2) is very close to the CBD1-CBD2 linker and the Ca3-Ca4 sites of CBD1 (either in NCX or CALX), thereby suggesting that the relevant interactions may affect the Ca folding ,123,124.2+-bound CALX1.1-CBD12 and CALX1.2-CBD12, it was suggested that slight differences in the interdomain angle (~8º) between the CBD1 and CBD2 domains determine different responses to regulatory Ca2+ in full-size CALX1.1 and CALX1.2 variants [2+-bound CBD12 is nearly identical for NCX1-CBD12-AD (117.4°) and CALX1.1-CBD12 (117.7°), which means that the fixed-angle alignment of CBDs cannot account for Ca2+-dependent activation (NCXs) and inhibition (CALX1.1) [2+-dependent activation of mammalian NCXs. According to this unifying mechanism, the Ca2+-dependent activation of mammalian NCXs involves the Ca2+-dependent tethering of two CBDs (due to the Ca2+ occlusion), which restricts the interdomain movements of CBDs to keep mammalian NCX active. Subsequently, a slow dissociation of occluded Ca2+ leads to NCX inactivation, where the time scale of NCX activation depends on the kinetics of the occluded Ca2+ dissociation. Even though the interdomain network of R532, D499, and D500 residues is a common structural module for the Ca2+ occlusion and subsequent tethering of CBDs either in NCX or CALX  than in NCXs (0.03–0.5 s−1) [2+ tethering could be related to translational and rotational movements of CBDs, remains to be resolved (see below).Based on the crystal structures of Cavariants . HoweverCALX1.1) . CumulatCALX1.1) ,180,181,CBD12-AD .4° and CCALX1.1) ,120,121,CALX1.1) ,123,124  or CALX , the strvariants ,123,124.CBD12-AD .4° and C0.5 s−1) ,123,124.2+ shifts the fractional distribution of conformational states toward a more constrained population [2+ binding rigidifies the interdomain movements of CBDs, where the average distance between the CBD1 and CBD2  remains unaffected. These findings are consistent with the population shift mechanism, according to which the ligand (Ca2+) binding to a given protein does not generate new conformational states; instead, the ion binding shifts a predominant population of unstable conformational states to a new population of more stable conformational states [2+ binding, the fraction of more rigid (constrained) conformational states becomes dominant at dynamic equilibrium. The unique feature of the population shift mechanism is that it avoids large conformational transitions requiring sizable free energy changes [2+-dependent regulation of NCXs since the quick and effective response of tissue-specific NCX variants is required to match the Ca2+-dependent events that occur from the millisecond time range to minutes and hours [The analyses of CALX1.1-CBD12, CALX1.2-CBD12, NCX1-CBD12, NCX2-CBD12, and NCX3-CBD12 by SAXS have shown that the occupation of the Ca3-Ca4 sites by Capulation ,120,121.l states ,189,190. changes ,185,190. changes ,184,185.nd hours ,189.2+ binding to the Ca3–Ca4 sites of NCX1-CBD12 or CALX-CBD12 restricts the linkers’ flexibility and interdomain movements of CBDs [2+ occlusion at Ca3-Ca4 of NCX and CALX rigidifies the backbone dynamics of the two-domain interface, which is coupled with CBD tethering through Ca2+ occlusion [2+-bound forms in all tested variants , although the Ca2+ binding narrows and shifts the population of conformational states under a dynamic equilibrium [2+-dependent tethering [Consistent with the population shift mechanism, NMR analysis revealed that Ca of CBDs ,180,181.cclusion ,123,124.ilibrium ,118. Altethering ,42, the ethering ,123,124.2+-dependent tethering of CBDs rigidifies the CBD interface in NCX and CALX [2+-dependent rigidification characteristically differs in NCX1-CBD12 (AD), NCX2-CBD12, NCX3-CBD12 (BC), and CALX1.1. These differences in the backbone rigidification are especially prominent when comparing NCX and CALX variants. In NCX1-CBD12-AD, the Ca2+ binding to CBD1 rigidifies the backbone from the Ca3–Ca4 sites of CBD1 up to the C-terminal tip of CBD2  conformational transitions. The mutually exclusive exons, A and B, are located at the C-terminus tip of CBD2 (on the loops that are directly involved in Ca2+ coordination) [2+ affinity at the Ca3-Ca4 sites of CBD1 (while decelerating the off-rates of occluded Ca2+ dissociation from Ca3-Ca4), whereas exon B structurally prevents Ca2+ binding to CBD2 . Thus, the mutually exclusive exons (A and B) not only condition the Ca2+ binding stoichiometry to CBD2 but also shape the Ca2+ affinity to CBD1 and CBD2 domains [Conceptually, the splicing segments of proteins contain intrinsically disordered regions, which avoids the formation of stable tertiary structures ,186,187.ination) ,40,41,42ination) ,123,124. to CBD2 A–C. In N at CBD1 B,C. In N domains ,121,122.2+ binding sites at CBD1 as well as slows down the dissociation of occluded Ca2+ from the Ca3-Ca4 sites [2+ binding sites of CBD1. Most probably, the exon-dependent rigidification of the CBD2 BC-loop and interdomain linker (upon Ca2+ binding to the Ca3-Ca4 sites of CBD1) reduces the translational movement of CBD12. In contrast, the exon-dependent rigidification of the neighboring β-strands in CBD2 might restrict the rotational motion of CBD2. Besides the exons’ compositions, the intrinsic folding energy of CBD2 may affect the integration of transitional and rotational movements. For example, the intrinsic structure of CBD2 might be more rigid in CALX than in NCXs, which may limit the rotational movements of CBD2, thereby causing a negative or no response to Ca2+ in CALX1.1 or CALX1.2, respectively.The cassette exons  are located at the unfolded segment of the FG-loop while being in proximity with the high-affinity sites Ca3-Ca4) of CBD1 and the interdomain CBD1-CBD2 linker ,40,41,42-Ca4 of Ca4 sites ,122,123.+-dependent inactivation [2 binding to XIP (auto-inhibitory domain) activates NCX1.1, whereas the Na+-dependent release of PIP2 from XIP inhibits NCX [2, a positively charged XIP helix (located on the TM5) anchors a negatively charged helix (situated on the CBD2-TM6 segment). According to this proposal, inhibitory  and activating  ligands can shift between the steady-state equilibrium between the active and inactive states, thereby characteristically shaping the outcomes of regulatory effects in distinct isoform/splice variants [Negatively charged phospholipids ,193, anitivation ,198,199.variants ,174,200 variants ,201.Another factor that may affect lipid–protein interactions is the varying composition of exons in the alternative splicing segment. For example, fatty acids more effectively inhibit NCX1.3 (BD), expressed in smooth muscle and kidney, than in the cardiac NCX1.1 (ACDEF) variant . These d4-fold differences in the transport rates observed between NCX1.1 and NCX_Mj [In contrast with mammalian NCXs, prokaryotic NCX_Mj is insensitive to varying compositions of lipids . Since nd NCX_Mj ,59,60,672+-dependent cleavage by proteases  among many others . Besidesee ref. ,157,158,see ref. .2) modulate dynamic features of the TM1/TM6 movements, where the associated altering in the OF/IF swapping limits the transport rates. Future experiments using the advanced approaches of Cryo-EM and HDX-MS techniques may address these issues by examining the nanodisc-reconstituted full-size variants of mammalian NCXs.Palmitoylation is a covalent addition of C16 fatty acids to protein cysteines via S-acylation, which controls the protein’s trafficking and insertion into the membrane while contributing to cellular signaling ,161,162.During the last decade, remarkable progress has been achieved toward the understanding of structure-dynamic mechanisms underlying the ion transport and regulation of NCX orthologs/isoforms/splice variants. This was achieved by applying especially suited multidisciplinary approaches for systematic analyses of the archaeal (NCX_Mj), invertebrate (CALX1–2), and mammalian (NCX1–3) sodium–calcium exchanger proteins , derived from eukaryotic NCXs and their analyses using SAXS, FRET, and HDX-MS, provided very useful information on the structure-dynamic basis of regulatory divergence in eukaryotic NCXs. Despite this progress, the isolated regulatory domains cannot be explored for studying the remote allosteric interactions between the regulatory and ion-transporting domains. Thus, the emerging goal is to explore new experimental systems and approaches to elucidate how the regulatory massages become diverged, transferred, and integrated in tissue-specific NCX variants. This underscores an urgent need for the discovery of the full-size structure of eukaryotic NCX. Most probably, this will be achieved using the advanced techniques of Cryo-EM. In conjunction with the full-size structure of mammalian NCX, the advanced approaches of the CRISPR/Cas9 techniques can be applied for elucidating the function and regulation of tissue-specific NCX variants under physiological and pathophysiological conditions. Once the full-size structure of mammalian NCX becomes available, this can provide new opportunities for structure-based devising of drug-like ligands for selective inhibition or activation of tissue-specific NCX isoform/splice variants. In the long-term run, this may identify drug candidates, which could be considered for focused clinical trials.


Whether GPCRs support the sensing of temperature as well as other chemical and physical modalities is not well understood.Introduction: Extracellular Ca2+ concentration (Ca2+o) modulates core body temperature and the firing rates of temperature-sensitive CNS neurons, and hypocalcemia provokes childhood seizures. However, it is not known whether these phenomena are mediated by Ca2+o-sensing GPCRs, including the calcium-sensing receptor (CaSR). In favor of the hypothesis, CaSRs are expressed in hypothalamic regions that support core temperature regulation, and autosomal dominant hypocalcemia, due to CaSR activating mutations, is associated with childhood seizures.Methods: Herein, we tested whether CaSR-dependent signaling is temperature sensitive using an established model system, CaSR-expressing HEK-293 cells.Results: We found that the frequency of Ca2+o-induced Ca2+i oscillations but not the integrated response was linearly dependent on temperature in a pathophysiologically relevant range. Chimeric receptor analysis showed that the receptor’s C-terminus is required for temperature-dependent modulation and experiments with the PKC inhibitor GF109203X and CaSR mutants T888A and T888M, which eliminate a key phosphorylation site, demonstrated the importance of repetitive phosphorylation and dephosphorylation.Discussion and Conclusion: CaSRs mediate temperature-sensing and the mechanism, dependent upon repetitive phosphorylation and dephosphorylation, suggests that GPCRs more generally contribute to temperature-sensing. It is a2+ , l-aminono acids  and glutno acids , inhibitno acids , and keyno acids . It is nno acids  and by tno acids .2+o concentration, the CaSR mediates oscillations in cytoplasmic free Ca2+ (Ca2+i) in various cell types including transfected HEK-293 cells that translate CaSR mRNA (2+o induces low frequency (1—4 min−1) Ca2+i oscillations  that arllations . HEK-293llations .2+i oscillations was sensitive to a large change in temperature in CaSR-expressing HEK-293 cells  human CaSR cDNA was cloned between the Kpn I and Xba I sites of pcDNA3.1 (+). All mutants were generated in pcDNA3.1(+)-WT CaSR. The Quikchange II site-directed mutagenesis protocol was used to introduce the point mutations T888A and T888M, as described previously : brieflyThe hCaSR/rmGluR1/rmGluR1 chimeric receptor construct was described previously . It encoThe identities of all mutants were confirmed by DNA sequencing .HEK-293 cells stably expressing the human CaSR (HEK-CaSR cells) or the hCaSR/rmGluR1/rmGluR1 (HEK-C/G/G) chimeric receptor were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) as described previously . Cells wFor experiments with transiently expressing CaSR constructs, when the cells had reached 85%–95% confluency they were transfected with XtremeGENE HP transfection reagent : briefly, 0.5 μg samples of WT or mutant DNA and 3 μL of transfection reagent were diluted with 100 μL of DMEM and allowed to complex at RT for 15 min. The transfection solution was added to the cell cultures to a final concentration of 9.1% (v/v).2, 20 mM HEPES-NaOH (pH 7.45) that contained 1 mM CaCl2, 0.8 mM NaH2PO4, and 1 mg ml−1 BSA for 2 h at 37°C. The cells were stored in loading solution prior to experiments. Fura-2-loaded HEK-CaSR cells were then placed in a perifusion chamber in the light path of a Zeiss Axiovert 200M fluorescence microscope (×63 objective) and perifused with PSS containing various concentrations of Ca2+o. The PKC inhibitor GF109203X (Sigma-Aldrich) was used at a final concentration of 250 nM.HEK-CaSR, HEK-C/G/G or transiently transfected HEK-293 cells were cultured on coverslips in 24-well plates and then loaded in the dark with Fura-2AM (5 μM) in physiological saline solution (PSS): 125 mM NaCl, 4 mM KCl, 0.1% (w/v) D-glucose, 1 mM MgClFura-2-loaded HEK-CaSR cells were excited using a Lambda DG-4 150 Watt xenon light source (Sutter Instruments), with alternating wavelengths of 340 and 380 nm at 0.5s intervals, and the fluorescent cells were imaged at 510 nm. Each data set consisted of 10 regions of interest each containing an individual cell and digital images were captured using an AxioCam camera controlled by Stallion SB.4.1.0 PC software (Carl Zeiss).via a peristaltic pump (Gilson Minipuls 2) and; 2) a Warner TC-344B Dual Channel Heat Controller that controlled the temperature of the microscope stage. To prevent heat losses, the inlet tubing to the microscope chamber was passed through a wide-bore 1.5 cm diameter tube containing water of the desired temperature that re-circulated via the external circuit of a thermostatically controlled water-bath. Using this system, the required perifusion solution temperatures were achieved by adjusting the demand temperatures of the water bath and the microscope stage as demonstrated by the temperatures measured in the effluent solutions (The temperature in the perifusion chamber of the fluorescent microscope and therefore of fura-2-loaded cells was set by 1) a recirculating heat exchange system that delivered solutions of the desired temperature into the chamber olutions . When th2+ 1.2 mM) and transported to the laboratory on ice. After isolation and washing to remove collagenase by centrifugation , aliquots of cells  were loaded into columns to lie above a layer of Biogel P4 (Biorad #1504124) and below a layer of Sephadex G-25 medium (Sigma-Aldrich #G-25150) as described previously and perifused with physiological saline solution  that contained bovine serum albumin 1 mg/ml  and various Ca2+ concentrations at 37°C as required. Perifusion columns were housed in metal holders that enabled full immersion and connections to be established upstream to the perifusion solutions and downstream to a peristaltic pump. The metal holders also protected the columns during transfer between waterbaths (see below). Samples were collected on ice and transferred to dry ice prior to storage at -80°C. Analysis for PTH was subsequently performed on a Siemens Immulite autoanalyzer . To permit studies of the effect of temperature on Ca2+o-regulated PTH secretion, two water baths (one equilibrated at 35°C and one equilibrated at 38°C) were placed in close proximity and identical pre-equilibrated PSS-albumin solutions  were immersed in both baths to allow prompt temperature exchange (∼30 s) when required. To enable temperature changeovers, the perifusion pump was stopped briefly (20–30s) and perifusion columns were transferred to the neighboring bath at the new temperature (38°C or back to 35°C) where its upstream connections were established to the new temperature pre-equilibrated solution, and the pump restarted.Human parathyroid cells were prepared by collagenase digestion of surgical specimens of normal parathyroid tissue as described previously  accordin2+i mobilization data consisted of excitation ratios (F340/F380) plotted against experiment time (min). Where indicated, Ca2+i responses from single cells were integrated with respect to time according to the ‘trapezoidal rule’, using the following equation:x is time interval between measurements (0.5 s) and y is F340/F380, to provide results in the form of integrated response units (IRUs) that were corrected for the baseline observed at control Ca2+o (0.5 mM), as described previously  for comparisons between regression lines.Statistical analyses were performed in GraphPad Prism version 8.0 using: Student’s unpaired 2+o (0.5 mM) and then exposed to an abrupt step-wise increase in Ca2+o to a near-maximally effective Ca2+o concentration, 5.0 mM. Once stable Ca2+i oscillations had been established, the temperature was lowered to 14°C for 10–15 min and subsequently restored to the baseline temperature (34°C). At baseline temperature (34°C) and at 5.0 mM Ca2+o, the Ca2+o-induced Ca2+i frequency was around 3 min−1  at low-baseline Ca 3 min−1 . As the  to 34°C . Thus, a2+o from 0.5 mM to 4.0 mM. These Ca2+o levels were selected based on the known Ca2+o concentration-response behaviour of HEK-CaSR cells  and the data were pooled.We next determined the effects of stepwise changes in temperature in the pathophysiological range 31°C–41°C on fura-2 loaded HEK-CaSR cells that were exposed to increases in CaSR cells . The expSR cells ; and 2) SR cells . For the2+o concentration (0.5 mM) all cells exhibited stable baseline Ca2+i levels. When Ca2+o was increased to 4.0 mM, however, around 95% of HEK-CaSR cells exhibited prompt oscillations in Ca2+i across the temperature range 31°C–41°C. At normal physiological temperature (37°C) the Ca2+i oscillation frequency was 4.21 ± 0.13 min−1 consistent with previous reports , and L-Phe increased Ca2+i oscillation frequency at all temperatures tested, without changing the slope of the temperature—Ca2+i frequency relationship  emulating the responses observed in HEK-CaSR cells, in which the CaSR was expressed stably , or with T888A was 1.03 ± 0.26 min−1 .In HEK-293 cells that were transiently transfected with the activating mutations T888A  or T888M2+o from 0.5 to 4.0 mM induced Ca2+i oscillations in around 65% of cells  and this response was independent of temperature over the range 31°C–41°C. Similar results were obtained with HEK-293 cells that were transiently transfected with T888A. Thus, temperature had a positive effect on the frequency of Ca2+o-induced Ca2+i oscillations in HEK-293 cells transiently transfected with the wild-type CaSR but had no effect on Ca2+i frequency in HEK-293 cells transiently transfected with either T888M or T888A .In cells that had been transiently transfected with T888M, an increase in Caor T888A . In part2+o-induced Ca2+i oscillations to sustained elevations in Ca2+i in HEK-CaSR cells (2+o at 37°C induced Ca2+i oscillations in approximately 95% of cells as observed previously and GF109203X (250 nM) transformed the Ca2+i responses from stable oscillations to sustained increases in ∼60% of cells, presumably as a consequence of the unopposed action of protein phosphatase activity  during the control period .Since phosphorylation of CaSR residue T888 is dependent on PKC , we nextSR cells . Exposuractivity  .The CaSR is a key multi-modal sensor that mediates responses to multiple metabolic signals including activating divalent cations Ca, and pH . It is a, and pH  and inor, and pH . In the 2+o-induced low frequency Ca2+i spikes (<1 min−1) that were insensitive to temperature  chimeric receptor, which is composed of the human CaSR’s VFT domain fused to the rat mGluR1α cysteine-rich domain, heptahelical domain and intracellular C-terminus, exhibited high Caperature . Althougperature  mGluR1-mllations . The resllations  and impl2+i oscillations (2+i and 2) CaSR-mediated suppression of PTH secretion -induced Ca2+i oscillations at 37°C and Ca2+i frequency was not modulated by temperature  negatively modulates core temperature in various species  and in hildhood , presuma2+o was raised from 0.8 mM to 1.2 mM. Conversely, when the temperature was raised from 35°C to 38°C, we observed enhanced PTH secretion at a Ca2+o concentration of 0.8 mM, together with enhanced suppression of PTH secretion at a Ca2+o concentration of 1.1 mM. However, the cell preparation failed to recover to its baseline PTH secretory rate upon return to the control temperature . Although the results are consistent with the temperature-sensing properties we observed in the present study, the failure of the parathyroid cell preparation to recover normal secretory control following large excursions in temperature leads us to conclude that animal rather than cell models are likely to be more informative. One such model would be a tissue-selective CaSR-null mouse in which upon deletion of the CaSR from the anterior preoptic nucleus, loss of Ca2+o-dependent negative modulation of core temperature would be expected.In considering appropriate models for investigating the pathophysiological significance of our findings, in preliminary experiments we screened a human parathyroid cell preparation, which expresses the CaSR endogenously, for secretory responses to temperature. We observed that switching from physiological temperature (37°C) to a low temperature (30°C) reversibly lowered PTH secretion, as expected for enzyme-dependent exocytosis from membrane-bound vesicles, and also exhibited an apparent, low temperature-dependent impairment of the CaSR-mediated inhibitory response, as Cathree key conditions  the nditions  (Criterinditions )  > 5 (warm sensitive) or <0.2 (cold sensitive)  . Furtherround 20 . Whetherround 20 , is co-eround 20 , as wellround 20 .The identification of the CaSR’s temperature-sensing properties extends its repertoire of physical and chemical sensing properties (reviews: . The res2+i oscillation frequency, and these responses were supported by PKC-dependent phosphorylation and, presumably, dephosphorylation of the CaSR C-terminal residue T888. Expression of the CaSR in thermoregulatory organs of the hypothalamus, observations that Ca2+o negatively modulates core body temperature together with the firing rates of thermoregulatory neurons, the existence of interactions between Ca2+o and the CaSR on the behavior of thermoregulatory ion channels, and the recognised impact of genetic disorders of calcium metabolism and/or the CaSR on thermoregulation and CNS function are all consistent with the hypothesis that the CaSR modulates thermal regulation in vivo.In conclusion, we have demonstrated that the CaSR responds to changes in temperature in the pathophysiological range 31°C–41°C. Temperature positively and reversibly modulated Ca


Chronic spontaneous urticaria (CSU) considerably alters patients’ quality of life, often for extended periods, due to pruriginous skin lesions, impaired sleep, unexpected development of angioedema, and failure of conventional treatments in properly controlling signs and symptoms. Recent research focused on the development of new therapeutic agents with higher efficacy. Although the production of specific immunoglobulin E (IgE) antibodies against certain allergens is not a characteristic of the disease, treatment with omalizumab, a monoclonal anti-IgE antibody, proved efficient and safe in patients with moderate to severe chronic spontaneous urticaria uncontrolled by H1-antihistamines. Ligelizumab, a high-affinity monoclonal anti-IgE antibody, may also efficiently relieve symptoms of unresponsive chronic urticaria to standard therapies. This comprehensive review aims to present recently acquired knowledge on managing chronic spontaneous urticaria with new anti-IgE antibodies. We conducted extensive research on the main databases  with no restrictions on the years covered, using the search terms “anti-IgE antibodies”, “omalizumab”, “ligelizumab”, and “chronic spontaneous urticaria”. The inclusion criteria were English written articles, and the exclusion criteria were animal-related studies. ClinicalTrials.gov was also reviewed for recent relevant clinical trials related to CSU treatment. CSU is a challenging disease with a significant effect on patients’ quality of life. Current therapies often fail to control signs and symptoms, and additional treatment is needed. New biologic therapies against IgE antibodies and FcεRIα receptors are currently under investigation in advanced clinical trials. We reviewed recently published data on CSU management using these novel treatments. The development of new and improved treatments for CSU will lead to a more personalized therapeutical approach for patients and provide guidance for physicians in better understanding disease mechanisms. However, some agents are still in clinical trials, and more research is needed to establish the safety and efficacy of these treatments. Chronic idiopathic urticaria or chronic spontaneous urticaria (CSU) is a debilitating disease that significantly impacts the quality of life. It is characterized by the development of wheals (hives), associated or not with angioedema for a period longer than 6 weeks, due to known or unknown apparent cause ,2. WhealUrticaria is considered a disease driven mainly by mast cells . SymptomBased on studies by Babaie et al. and Grieco et al. IL-6 plays a significant role in the pathogenesis of chronic urticaria by promoting the trans-signaling capacity during the inflammatory response . IL-6 isProduction of IgE plays a central role in the pathogenesis of allergic diseases. IgE is present both in the peripheral circulation and on the surface of different cell types, bound to low-affinity receptors, CD23 and high-affinity receptors, FcεRIα . The binThis comprehensive review aims to present recently acquired knowledge on managing chronic spontaneous urticaria with new anti-IgE antibodies.This non-systematic review aims to clarify the known data regarding anti-IgE biological therapy used for treating chronic spontaneous urticaria. We conducted extensive research on the main databases  with no restrictions on the years covered, using the search terms “anti-IgE antibodies”, “omalizumab”, “ligelizumab”, and “chronic spontaneous urticaria”. The inclusion criteria were English written articles, and the exclusion criteria were animal-related articles. A total of 38 studies were included for review. We identified 22 studies containing the terms “omalizumab” and “anti-IgE antibodies”, and 16 articles containing the term “ligelizumab” between the years 2014–2022. ClinicalTrials.gov was also reviewed for recent relevant clinical trials related to CSU treatment.Anti-IgE therapy;Management of chronic spontaneous urticaria with omalizumab and ligelizumab in clinical practice;Potential biomarkers for the effectiveness of chronic urticaria treatment.Chronic idiopathic urticaria or chronic spontaneous urticaria is a very challenging and frustrating disease for both patients and physicians. Consequently, different biological therapies have been developed as conventional treatments often fail to control the signs and symptoms . During The first licensed biological treatment for chronic spontaneous urticaria is the monoclonal anti-IgE antibody, omalizumab. It was approved for the treatment of mild to severe and long-lasting allergic asthma in 2003. In 2014, it was approved for the treatment of spontaneous urticaria for patients 12 years and older at a dose of 150 mg every 4 weeks and 300 mg every 4 weeks. This treatment primarily aims to achieve the relief of symptoms, such as pruritus and pain, as well as complete disappearance of the clinical signs in the least possible time frame. In addition, the treatment aims to ensure a better quality of life for chronic urticaria patients [2LEN/EuroGuiDerm/APAAACI guidelines for the management of chronic spontaneous urticaria, a four-step treatment algorithm is recommended , a long-acting IgE trap-Fc fusion protein that may exhibit higher and more durable binding to IgE compared with omalizumab . HoweverAt present, few questions still remain unanswered. The discrepancies between clinical trial results and those seen in everyday practice are vital for practitioners. For example, in some instances, a better response to omalizumab has been observed in real-life clinical settings compared with pivotal randomized controlled trials ,15,16.Real-life practical evidence has demonstrated that patients with chronic spontaneous urticaria may exhibit a rapid response to omalizumab. A retrospective evaluation of patients who underwent treatment with omalizumab outside of clinical trials showed a quick control of the symptoms, with 57% of patients achieving complete clinical remission within a week . In addiIn another retrospective study conducted in Spain on 110 patients with incompletely controlled chronic urticaria, the administration of omalizumab resulted in a complete or significant response in 82% of the patients. Moreover, 6% of patients were able to discontinue the other administered drugs for chronic urticaria and remained free of symptoms under monotherapy with omalizumab . After a2LEN/EuroGuiDerm/APAAACI guidelines for the management of chronic spontaneous urticaria, the recommended initial dose is 300 mg every 4 weeks. Dosing is independent of total serum IgE [In accordance with the current international EAACI/GAerum IgE . Patienterum IgE . PatientLigelizumab, at a dose of 240 mg every 4 weeks, safely and effectively controls not only chronic spontaneous urticaria, but also other chronic urticaria subtypes, such as delayed pressure urticaria , solar uIn a comparison study between ligelizumab and omalizumab performed by Soong W. et al. it was noticed that ligelizumab has the ability to inhibit IgE binding to FcεRIα more effectively compared with CD23, thus being more efficient than omalizumab . MoreoveRecent data suggest that ligelizumab’s superiority may be due to differences in their IgE binding sites . Since oIn conclusion, preliminary clinical data and recent reports demonstrate more prolonged suppression of IgE with ligelizumab compared with omalizumab.The prediction of variations in the disease activity in response to treatment can be very helpful in improving treatment algorithms. Therefore, identifying biomarkers that can be used to predict the course of the disease and the extent of variation in the activity of chronic urticaria upon administration of a particular treatment would represent a significant advancement in providing an optimal individualized therapeutic approach. This is applicable for all the drugs currently used to treat chronic urticaria, such as omalizumab, leukotriene receptor antagonists, cyclosporine, and H1-antihistamines.Among patients treated with cyclosporine, a positive serum basophil histamine release assay suggests that these patients are more inclined to respond to cyclosporine treatment than patients with a negative test .For the moment, data on which and how biomarkers may anticipate the result of the other treatments are lacking, but research on the subject is ongoing. An example of the interest in the field is a recent retrospective study that included 41 patients with chronic refractory urticaria and concluded that the absence of basophil CD203c upregulation activity in the patients’ serum was correlated with the clinical response to omalizumab . The uprGrieco et al. discovered higher levels of IL-6 and IFN-gamma in patients with chronic spontaneous urticaria compared with those in the control group [In support with the perspective described above, biomarkers require further evaluation in prospective studies due to the strong potential to be valuable, clinically significant, response indicators to specific treatments.Several other potential therapies for chronic spontaneous urticaria are currently under investigation in phases 1, 2, 3 and 4 clinical trials.Monoclonal antibodies against inflammatory cytokines released by mast cells, such as interleukin-5 (IL-5), the IL-5 receptor (IL-5R) or IL-4R have turned into recent research targets for CSU treatment . In addiIn recent years, new approaches in disrupting pro-inflammatory mechanisms have been explored. It was observed that the total number of mast cells could be depleted by targeting the stem cell growth factor receptor-tyrosine kinase KIT . A new gDue to the potential side effects and immunoreactivity , these pChronic spontaneous urticaria has a significant medical impact worldwide due to its increasing prevalence in a general, age-dependent population, the potential to associate systemic symptoms and life-threatening angioedema, and due to the unpredictable evolution, particularly in patients with uncontrolled disease. Therefore, extending the therapeutical possibilities in monotherapy or integrative approaches is a priority. In the last decades, research focused on developing monoclonal anti-IgE antibodies. The first monoclonal anti-IgE antibody was omalizumab, which is now recommended by EAACI guidelines for patients with refractory urticaria. A second monoclonal anti-IgE antibody is ligelizumab, possibly with a higher IgE affinity than omalizumab. Additional recent agents are still waiting for evaluation by several ongoing trials. Although progress has been noted through the addition of novel therapeutic approaches, we believe that more insight and research are required on IgE antibody modulation correlated with the clinical response of CSU. Certainly, new biologic therapies and possible predictive biomarkers will be available in the future, allowing medical professionals to prescribe innovative and better treatments for patients struggling with chronic urticaria.