Store-operated Calcium Entry Channels Research Articles

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases like thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 GPCRs-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and TRPC3 channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells' contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.

Homoplantaginin attenuates high glucose-induced vascular endothelial dysfunction via inhibiting store-operated calcium entry channel and endoplasmic reticulum stress.

The activation of store-operated calcium entry (SOCE) channel and endoplasmic reticulum stress (ERS) induced by high glucose (HG) is recognized as a major cause of vascular endothelial dysfunction. This study aims to investigate the protective effect of homoplantaginin (Hom) on HG-induced endothelial dysfunction. HG-induced vascular endothelial dysfunction model in human umbilical vein endothelial cells (HUVECs) and rat-isolated thoracic aortas were established to observe the protective effect of Hom, further evaluated the mechanism of SOCE channel and ERS in the pathogenesis. Hom increased the levels of nitric oxide (NO) and phospho-endothelial nitric oxide synthase (p-eNOS) in HUVECs and isolated rat thoracic aortas in a dose-dependent manner, restored acetylcholine-mediated endothelium-dependent vasodilation. Network pharmacology showed that the pathogenesis of diabetic vascular complications may involve calcium (Ca2+) signal pathway. Hom reduced Ca2+ concentration via blocking SOCE channel in HUVECs, and resisted ERS activation by down-regulating ERS-related proteins expression. Importantly, SKF96365 (SOCE inhibitor) intervention experiment showed that Hom inhibited ERS activation by blocking the SOCE channel, further increased the levels of NO and p-eNOS. Hom could alleviate HG-induced vascular endothelial dysfunction by inhibiting SOCE channel and ERS. This provided a potential pharmacological intervention strategy for the treatment of vascular endothelial dysfunction.

β-carotene targets IP3R/GRP75/VDAC1-MCU axis to renovate LPS-induced mitochondrial oxidative damage by regulating STIM1

Endoplasmic reticulum (ER) and mitochondria are the main sites for the storage and regulation of Ca2+ homeostasis. An imbalance of Ca2+ homeostasis can cause ER stress and mitochondrial dysfunction, thereby inducing apoptosis. The store-operated calcium entry (SOCE) is the main channel for extracellular calcium influx. Mitochondria-associated endoplasmic reticulum (MAM) is an important agent for Ca2+ transfer from the ER to the mitochondria. Therefore, regulation of SOCE and MAMs has potential therapeutic value for disease prevention and treatment. In this study, bovine mammary epithelial cells (BMECs) and mice were used as models to explore the mechanisms of β-carotene to relieve ER stress and mitochondrial dysfunction. BAPTA-AM, EGTA (Ca2+ inhibitor), and BTP2 (SOCE channel inhibitor) alleviated ER stress and mitochondrial oxidative damage induced by increased intracellular Ca2+ levels after lipopolysaccharide (LPS) stimulation. Furthermore, inhibition of ER stress by 4-PBA (ER stress inhibitor), 2-APB (IP3R inhibitor), and ruthenium red (mitochondrial calcium uniporter (MCU) inhibitor) restored mitochondrial function by reducing mitochondrial ROS. Our data also confirm that β-carotene targeted STIM1 and IP3R channels to repair LPS-induced ER stress and mitochondrial disorders. Consistent with the in vitro study, in vito experiments in mice further showed that β-carotene attenuated LPS-induced ER stress and mitochondrial oxidative damage by inhibiting the expression of STIM1 and ORAI1, and reducing the level of Ca2+ in mouse mammary glands. Therefore, ER stress-mitochondrial oxidative damage mediated by the STIM1-ER-IP3R/GRP75/VDAC1-MCU axis plays an vital role in the development of mastitis. Our results provided novel ideas and therapeutic targets for the prevention and treatment of mastitis.

Pregnancy-Specific Glycoprotein 9 Enhances Store-Operated Calcium Entry and Nitric Oxide Release in Human Umbilical Vein Endothelial Cells

We explored changes in pregnancy-specific glycoprotein 9 (PSG9) levels in the serum of patients with preeclampsia and the effects and underlying mechanisms of PSG9 effects on calcium (Ca2+) homeostasis and nitric oxide (NO) release in human umbilical vein endothelial cells (HUVECs). Western blotting was used to detect protein expression levels, and an NO fluorescence probe was used to examine NO production. Intracellular Ca2+ concentrations were measured using a Ca2+-sensitive fluorescent dye under a fluorescence microscope. Compared with those in healthy pregnant women, serum PSG9 levels were significantly decreased in patients with preeclampsia. PSG9 (0.1 μg/mL) treatment of HUVECs significantly enhanced the expression levels of store-operated calcium entry (SOCE) channel proteins Orai1 and Orai2, but not Orai3, and of endothelial nitric oxide synthase (eNOS) and NO production. Pretreatment with an inhibitor of SOCE (BTP2) abolished PSG9-enhanced Orai1, Orai2, and eNOS expression levels and NO production in HUVECs. The mechanisms underlying SOCE that were PSG9 enhanced in HUVECs appear to involve the Ca2+/eNOS/NO signaling pathway. These findings suggest that serum PSG9 levels may be a potential biomarker for monitoring the occurrence or development of preeclampsia in pregnancy and that PSG9 may be a potential therapeutic target for the treatment of preeclampsia.

Suppression of Store-operated Calcium Entry Channels and Cytokine Release by Cannabinoids.

Suppression of Store-operated Calcium Entry Channels and Cytokine Release by Cannabinoids.

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel-a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

PKC-\u03b2 modulates Ca2+ mobilization through Stim1 phosphorylation

BackgroundCalcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool.ObjectivesHere, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1.MethodsSeveral biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope.ResultsOur data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors.ConclusionIn sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

A high-concentrate diet provokes inflammation, endoplasmic reticulum stress, and apoptosis in mammary tissue of dairy cows through the upregulation of STIM1/ORAI1

High-concentrate feeding can induce subacute ruminal acidosis, which leads to mammary tissue injury in dairy cows. Therefore, the purpose of this research was to evaluate the effect of high-concentrate feeding on STIM1 (stromal interaction molecule 1)/ORAI1 (Orai calcium release-activated calcium modulator 1)-mediated inflammation, endoplasmic reticulum stress (ERS), and apoptosis in the mammary tissue of dairy cows. A total of 12 healthy mid-lactating Holstein cows of similar weight were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 6:4) and a low-concentrate (LC) group (concentrate:forage = 4:6). The trial lasted for 3 wk. After the feeding experiment, rumen fluid, lacteal vein blood, and mammary tissue samples were collected. The results showed that the HC diet significantly increased blood lipopolysaccharide levels, decreased ruminal pH, and upregulated the concentrations of Ca2+ and proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, and the enzyme activities of caspase-3, caspase-9, PKC, and IKK. The upregulation of STIM1, ORAI1, PKCα, IKKβ, phosphorylated-IκBα, phosphorylated-p65, TNF-α, and IL-1α proteins in the HC group indicated activation of the STIM1/ORAI1-mediated inflammatory signaling pathway compared with that in the LC group. The HC diet also induced ERS by increasing the mRNA and protein abundances of GRP78, CHOP, PERK, ATF6, and IRE1α in the mammary tissue. Compared with the LC group, the mRNA expression levels and protein abundances of caspase-3, cleaved caspase-3, caspase-9, and BAX were markedly increased in the HC group. However, the mRNA and protein expression levels of Bcl-2 were significantly decreased in the HC group. Therefore, this study demonstrated that the HC diet can activate the store-operated calcium entry channel by upregulating the expression of STIM1 and ORAI1 and induce inflammation, ERS, and apoptosis in the mammary tissue of dairy cows.

Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel.

Simple SummaryPancreatic cancer (PC) is one of the most lethal forms of cancers with 5-year mean survival rate of less than 10%. Most of the PC associated deaths are due to metastasis to secondary sites. Calcium (Ca2+) signaling plays a critical role in regulating hallmarks of cancer progression including cell proliferation, migration and apoptotic resistance. Here, we demonstrate that a highly Ca2+ selective plasma membrane channel Orai3 is overexpressed in PC and is associated with poor prognosis in PC patients. Our data demonstrate that Orai3 modulates PC cell proliferation, apoptosis and migration. We further reveal that Orai3 regulates PC metastasis in immune-compromised mice. Collectively, our study establishes Orai3 as an attractive therapeutic target for managing PC metastasis, which may lead to better prognosis.Store operated Ca2+ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous Ca2+ influx pathway. Although the role of Orai1 channels is well studied, the significance of Orai2/3 channels is still emerging in nature. In this study, we performed extensive bioinformatic analysis of publicly available datasets and observed that Orai3 expression is inversely associated with the mean survival time of PC patients. Orai3 expression analysis in a battery of PC cell lines corroborated its differential expression profile. We then carried out thorough Ca2+ imaging experiments in six PC cell lines and found that Orai3 forms a functional SOCE channel in PC cells. Our in vitro functional assays show that Orai3 regulates PC cell cycle progression, apoptosis and migration. Most importantly, our in vivo xenograft studies demonstrate a critical role of Orai3 in PC tumor growth and secondary metastasis. Mechanistically, Orai3 controls G1 phase progression, matrix metalloproteinase expression and epithelial-mesenchymal transition in PC cells. Taken together, this study for the first-time reports that Orai3 drives aggressive phenotypes of PC cells, i.e., migration in vitro and metastasis in vivo. Considering that Orai3 overexpression leads to poor prognosis in PC patients, it appears to be a highly attractive therapeutic target.

A New Muscle Activation Dynamics Model, That Simulates the Calcium Kinetics and Incorporates the Role of Store-Operated Calcium Entry Channels, to Enhance the Electromyography-Driven Hill-Type Models.

Hill-type models are frequently used in biomechanical simulations. They are attractive for their low computational cost and close relation to commonly measured musculotendon parameters. Still, more attention is needed to improve the activation dynamics of the model specifically because of the nonlinearity observed in the electromyography (EMG)-force relation. Moreover, one of the important and practical questions regarding the assessment of the model's performance is how adequately can the model simulate any fundamental type of human movement without modifying model parameters for different tasks? This paper tries to answer this question by proposing a simple physiologically based activation dynamics model. The model describes the kinetics of the calcium dynamics while activating and deactivating the muscle contraction process. Hence, it allowed simulating the recently discovered role of store-operated calcium entry (SOCE) channels as immediate counterflux to calcium loss across the tubular system during excitation-contraction coupling. By comparing the ability to fit experimental data without readjusting the parameters, the proposed model has proven to have more steady performance than phenomenologically based models through different submaximal isometric contraction levels. This model indicates that more physiological insights are key for improving Hill-type model performance.

Using a new muscle activation model to improve the stability of force estimation with Hill-type models over different activation levels

Hill-type models are attractive in biomechanics simulation due to their low computational cost and commonly measured parameters. They mainly consist of two parts, the activation dynamics, and the contraction dynamics. Since Hill-type models are phenomenological models, it is meant to capture the process’s main characteristics using a descriptive system that approximates the behaviour of the real physical one. This paper developed a novel activation dynamics formulation to estimate the force of the triceps surae muscles at two different contraction levels without retuning the model parameters. The new formulation simulates the cell calcium dynamics as well as the recently discovered role of store-operated calcium entry (SOCE) channels. The proposed model reflects the main characteristics required from the activation model, which are the electro-mechanical delay, the process nonlinearity, and the ability to couple the activation process with some parameters from the subsequent force-generating process. The parameters of the model are task-independent to closely approximate the muscle function and to have the ability to simulate different movements adequately. The model shows a more stable performance along with different contraction levels in comparison with two other activation models.

Calcium Sensors STIM1 and STIM2 Regulate Different Calcium Functions in Cultured Hippocampal Neurons.

There are growing indications for the involvement of calcium stores in the plastic properties of neurons and particularly in dendritic spines of central neurons. The store-operated calcium entry (SOCE) channels are assumed to be activated by the calcium sensor stromal interaction molecule (STIM)which leads to activation of its associated Orai channel. There are two STIM species, and the differential role of the two in SOCE is not entirely clear. In the present study, we were able to distinguish between transfected STIM1, which is more mobile primarily in young neurons, and STIM2 which is less mobile and more prominent in older neurons in culture. STIM1 mobility is associated with spontaneous calcium sparks, local transient rise in cytosolic [Ca2+]i, and in the formation and elongation of dendritic filopodia/spines. In contrast, STIM2 is associated with older neurons, where it is mobile and moves into dendritic spines primarily when cytosolic [Ca2+]i levels are reduced, apparently to activate resident Orai channels. These results highlight a role for STIM1 in the regulation of [Ca2+]i fluctuations associated with the formation of dendritic spines or filopodia in the developing neuron, whereas STIM2 is associated with the maintenance of calcium entry into stores in the adult neuron.

Orai-1 and Orai-2 regulate oral cancer cell migration and colonisation by suppressing Akt/mTOR/NF-κB signalling

Despite remarkable progress in understanding and treating oral cancer (OC), it still remains one of the life-threatening diseases and predominant cancers in the world. Therefore, deciphering the molecular mechanisms of this disease would help us to develop highly efficacious therapies. Multiple lines of evidence suggest that calcium and its dysregulation play significant role in the development of various cancers. As an adaptation of survival mechanism, upon depletion of ER calcium stores, store-operated calcium entry (SOCE) has been induced via SOCE channels (SOCC) in various mammalian cells. SOCC are regulated by Orai-1, Orai-2 and Orai-3 located on plasma membrane and two calcium-sensing ER membrane proteins known as stromal interaction molecules (STIM-1 and STIM-2). Hence, the present study was aimed at analysing the role of Orai-1 and Orai-2 in oral cancer and the underlying mechanism. Our results suggest that both Orai-1 and Orai-2 proteins were overexpressed in oral cancer tissues and cell lines (SAS) compared to normal epithelial tissues and cell lines respectively. In addition, silencing of Orai-1 and Orai-2 via chemical SOCE inhibitors and siRNAs inhibited calcium uptake and suppressed oral cancer cell proliferation, colony formation and migration. Furthermore, silencing of Orai-1 and Orai-2 inhibited Akt/mTOR/NF-κB pathway in oral cancer cells. Interestingly, tobacco carcinogen NNN and synthetic carcinogen 4-NQO, enhanced the expression of Orai-1 and Orai-2 in SAS cells. Therefore, we conclude that Orai-1 and Orai-2 have significant role in oral cancer and can be further explored to develop novel therapies for the treatment of this disease.

Calcium‐Dependent Localization of S100A6 in Pulmonary Microvascular Endothelial Cells

During inflammation, mediators increase calcium influx into endothelial cells through store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current ISOC promotes endothelial barrier disruption through the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased permeability. Identifying mechanisms of ISOC inhibition may aid in the development of therapeutic strategies against endothelial permeability‐associated vascular inflammation. Our studies indicate FK506‐binding protein 51 (FKBP51), in conjunction with protein phosphatase 5 (PPP5C), inhibit ISOC and calcium entry‐induced inter‐endothelial cell gap formation. Further, we determined that the calcium‐binding protein S100A6 is necessary for the PPP5C‐FKBP51‐mediated inhibition of ISOC in pulmonary microvascular endothelial cells (PMVECs). In PMVECs, S100A6 interacts with the ISOC channel in a calcium‐dependent manner, i.e., following an increase in cytosolic calcium. However, we do not know whether calcium entry specifically through the ISOC channel or through other SOCE channels that provides the calcium source to promote S100A6 interaction with the ISOC channel.MethodsPMVECs were used for co‐precipitation and immunocytochemistry‐based studies. When cells are treated with rolipram (R) plus thapsigargin (Tg), ISOC and other SOCE channels are activated. However, when cells are treated with Tg alone, ISOC is not activated, but other SOCE channels are activated. The TRPC4 subunit of the ISOC channel was immunoprecipitated and probed for co‐precipitation of S100A6.ResultsFollowing R/Tg treatment, robust co‐precipitation of S100A6 with TRPC4 was observed, whereas following Tg alone only minor co‐precipitation was seen. Immunocytochemistry revealed that in untreated cells S100A6 localization is principally cytosolic. Following R/Tg treatment, but not Tg alone, a distinct membrane‐delimited pool of S100A6 was observed, particularly at sites of cell‐cell contact. Membrane localization was confirmed by co‐localization with wheat germ agglutinin (WGA).ConclusionOverall, our data reveal that calcium entry specifically through the ISOC channel is an important determinant of S100A6 translocation to the plasma membrane promoting interaction of S100A6 with the ISOC channel.Support or Funding InformationNIH HL R56HL107778

The polyphenol ellagic acid exerts anti-inflammatory actions via disruption of store-operated calcium entry (SOCE) pathway activators and coupling mediators

Ellagic acid, a naturally occurring phenol found in a variety of fruits and nuts has been shown to possess anti-inflammatory properties. However, the mechanism of action behind its anti-inflammatory action is unclear. Using human Jurkat T cells, our study examined the effects of ellagic acid (EA) on Ca2+ handling, in particular, store-operated Ca2+ entry (SOCE), a process critical to proper T cell function. We observed that the acute addition of EA-induced Ca2+ release with an EC50 of 63 μM. The Ca2+ release was significantly attenuated by Xestospongin C, a known inhibitor of the Inositol 1,4,5-trisphosphate receptor (IP3R) channel and was unaffected by the phospholipase C (PLC) inhibitor, U73122. Furthermore, chronic incubation of Jurkat T cells with EA not only decreased the ATP-induced Ca2+ release but also diminished the SOCE-mediated Ca2+ influx in a dose-dependent manner. This inhibition was confirmed by reduced Mn2+ entry rates in the EA-treated cells. The ATP-induced Ca2+ entry was also attenuated in EA-treated HEK293 cells transiently transfected with SOCE channel Orai1-myc and ER-sensor stromal interaction molecule (STIM1) (HEKSTIM/Orai). Moreover, EA treatment interfered with the Orai1 and STIM1 coupling by disrupting STIM1 puncta formation in the HEKSTIM/Orai cells. We observed that EA treatment reduced cytokine secretion and nuclear factor of activated T-cell transcriptional activity in stimulated T cells. Hence, by inhibiting SOCE mediated Ca2+ influx, EA decreased downstream activation of pro-inflammatory mediators. These results suggest a novel target for EA-mediated effects and provide insight into the mechanisms underlying EA-mediated anti-inflammatory effects.

Calcium channel Orai1 promotes lymphocyte IL-17 expression and progressive kidney injury.

We hypothesized that the store-operated calcium entry (SOCE) channel, Orai1, participates in the activation of Th17 cells and influences renal injury. In rats, following renal ischemia/reperfusion (I/R), there was a rapid and sustained influx of Orai1+ CD4 T cells and IL-17 expression was restricted to Orai1+ cells. When kidney CD4+ cells of post-acute kidney injury (post-AKI) rats were stimulated with angiotensin II and elevated Na+ (10-7 M/170 mM) in vitro, there was an enhanced response in intracellular Ca2+ and IL-17 expression, which was blocked by SOCE inhibitors 2APB, YM58483/BTP2, or AnCoA4. In vivo, YM58483/BTP2 (1 mg/kg) attenuated IL-17+ cell activation, inflammation, and severity of AKI following either I/R or intramuscular glycerol injection. Rats treated with high-salt diet (5-9 weeks after I/R) manifested progressive disease indicated by enhanced inflammation, fibrosis, and impaired renal function. These responses were significantly attenuated by YM58483/BTP2. In peripheral blood of critically ill patients, Orai1+ cells were significantly elevated by approximately 10-fold and Th17 cells were elevated by approximately 4-fold in AKI versus non-AKI patients. Further, in vitro stimulation of CD4+ cells from AKI patients increased IL-17, which was blocked by SOCE inhibitors. These data suggest that Orai1 SOCE is a potential therapeutic target in AKI and CKD progression.

Calcium channel Orai1 promotes lymphocyte IL-17 expression and progressive kidney injury.

We hypothesized that the store-operated calcium entry (SOCE) channel, Orai1, participates in the activation of Th17 cells and influences renal injury. In rats, following renal ischemia/reperfusion (I/R), there was a rapid and sustained influx of Orai1+ CD4 T cells and IL-17 expression was restricted to Orai1+ cells. When kidney CD4+ cells of post-acute kidney injury (post-AKI) rats were stimulated with angiotensin II and elevated Na+ (10-7 M/170 mM) in vitro, there was an enhanced response in intracellular Ca2+ and IL-17 expression, which was blocked by SOCE inhibitors 2APB, YM58483/BTP2, or AnCoA4. In vivo, YM58483/BTP2 (1 mg/kg) attenuated IL-17+ cell activation, inflammation, and severity of AKI following either I/R or intramuscular glycerol injection. Rats treated with high-salt diet (5-9 weeks after I/R) manifested progressive disease indicated by enhanced inflammation, fibrosis, and impaired renal function. These responses were significantly attenuated by YM58483/BTP2. In peripheral blood of critically ill patients, Orai1+ cells were significantly elevated by approximately 10-fold and Th17 cells were elevated by approximately 4-fold in AKI versus non-AKI patients. Further, in vitro stimulation of CD4+ cells from AKI patients increased IL-17, which was blocked by SOCE inhibitors. These data suggest that Orai1 SOCE is a potential therapeutic target in AKI and CKD progression.

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca2+]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca2+]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.

Na-K-2Cl Cotransporter and Store-Operated Ca2+ Entry in Pacemaking by Interstitial Cells of Cajal

Pacemaker depolarization in interstitial cells of Cajal (ICCs) is believed to be induced by Ca2+ transients and activation of anoctamin-1 (Ano1) channels in the plasma membrane. However, block of store-operated calcium entry (SOCE) or the Na-K-2Cl cotransporter (NKCC1) terminates pacemaker activity in ICC, indicating these transporters are involved in the initiation or maintenance of pacemaker activity. We hypothesized that SOCE contributes to pacemaker depolarization by maintaining [Ca2+] in the endoplasmic reticulum, which is the underlying source of Ca2+ transients for activation of Ano1. NKCC1 maintains the Cl− gradient supporting the driving force for inward current mediated by Ano1. Currently mechanisms sustaining release of Ca2+ and activation of Ano1 channels during the plateau phase of slow waves are unknown, but the reverse mode of the Na+/Ca2+ exchange may contribute. We generated a mathematical model of pacemaker activity based on current empirical observations from ICC of mouse small intestine that incorporates functions of SOCE and NKCC1. This model reproduces experimental findings, suggesting roles for SOCE and Ano1 channels: blocking of either NKCC1 or SOCE in our model terminates pacemaker activity. Direct contribution of NKCC1 to pacemaker activity in a beat-to-beat manner is not predicted by our model. Instead, NKCC1 plays a maintenance role supporting the driving force for Cl− efflux. Incorporation of SOCE allows the model to drive pacemaker activity without a diastolic depolarization, as observed in cardiac pacemaking. Further biological experiments are necessary to validate and further refine the roles of NKCC1, Na+/Ca2+ exchange, and Ano1 in the pacemaker mechanism of ICC.

New insights into acute pancreatitis.

The incidence of acute pancreatitis continues to increase worldwide, and it is one of the most common gastrointestinal causes for hospital admission in the USA. In the past decade, substantial advancements have been made in our understanding of the pathophysiological mechanisms of acute pancreatitis. Studies have elucidated mechanisms of calcium-mediated acinar cell injury and death and the importance of store-operated calcium entry channels and mitochondrial permeability transition pores. The cytoprotective role of the unfolded protein response and autophagy in preventing sustained endoplasmic reticulum stress, apoptosis and necrosis has also been characterized, as has the central role of unsaturated fatty acids in causing pancreatic organ failure. Characterization of these pathways has led to the identification of potential molecular targets for future therapeutic trials. At the patient level, two classification systems have been developed to classify the severity of acute pancreatitis into prognostically meaningful groups, and several landmark clinical trials have informed management strategies in areas of nutritional support and interventions for infected pancreatic necrosis that have resulted in important changes to acute pancreatitis management paradigms. In this Review, we provide a summary of recent advances in acute pancreatitis with a special emphasis on pathophysiological mechanisms and clinical management of the disorder.