Intracellular Calcium Accumulation Research Articles

Beyond the Brain: The Systemic Pathophysiological Response to Acute Ischemic Stroke.

Stroke research has traditionally focused on the cerebral processes following ischemic brain injury, where oxygen and glucose deprivation incite prolonged activation of excitatory neurotransmitter receptors, intracellular calcium accumulation, inflammation, reactive oxygen species proliferation, and ultimately neuronal death. A recent growing body of evidence, however, points to far-reaching pathophysiological consequences of acute ischemic stroke. Shortly after stroke onset, peripheral immunodepression in conjunction with hyperstimulation of autonomic and neuroendocrine pathways and motor pathway impairment result in dysfunction of the respiratory, urinary, cardiovascular, gastrointestinal, musculoskeletal, and endocrine systems. These end organ abnormalities play a major role in the morbidity and mortality of acute ischemic stroke. Using a pathophysiology-based approach, this current review discusses the pathophysiological mechanisms following ischemic brain insult that result in end organ dysfunction. By characterizing stroke as a systemic disease, future research must consider bidirectional interactions between the brain and peripheral organs to inform treatment paradigms and develop effective, comprehensive therapeutics for acute ischemic stroke.

Role of Calcium Signaling in Prostate Cancer Progression: Effects on Cancer Hallmarks and Bone Metastatic Mechanisms.

Advanced prostate cancers that progress to tumor metastases are often considered incurable or difficult to treat. The etiology of prostate cancers is multi-factorial. Among other factors, de-regulation of calcium signals in prostate tumor cells mediates several pathological dysfunctions associated with tumor progression. Calcium plays a relevant role on tumor cell death, proliferation, motility-invasion and tumor metastasis. Calcium controls molecular factors and signaling pathways involved in the development of prostate cancer and its progression. Such factors and pathways include calcium channels and calcium-binding proteins. Nevertheless, the involvement of calcium signaling on prostate cancer predisposition for bone tropism has been relatively unexplored. In this regard, a diversity of mechanisms triggers transient accumulation of intracellular calcium in prostate cancer cells, potentially favoring bone metastases development. New therapies for the treatment of prostate cancer include compounds characterized by potent and specific actions that target calcium channels/transporters or pumps. These novel drugs for prostate cancer treatment encompass calcium-ATPase inhibitors, voltage-gated calcium channel inhibitors, transient receptor potential (TRP) channel regulators or Orai inhibitors. This review details the latest results that have evaluated the relationship between calcium signaling and progression of prostate cancer, as well as potential therapies aiming to modulate calcium signaling in prostate tumor progression.

Enhanced thermal stability of lactic acid bacteria during spray drying by intracellular accumulation of calcium

Lactococcus lactis ssp. cremoris and Lactobacillus acidophilus NCFM were cultured in growth media supplemented with CaCl2 to induce intracellular accumulation of Ca2+. Both strains grown with 10 mM Ca2+ demonstrated enhanced thermal stability, with increased survival after spray drying from 49.9% to 64.9% for the coccus and from 35.5% to 43.3% for the rod. The increased intracellular Ca2+ level was confirmed with murexide assay. Adding Ca2+ in the reconstitution solution could not improve the viability of spray dried cells at extended storage stages. We hypothesized that the over-presence of intracellular Ca2+ might stimulate the activity of heat shock proteins, increasing cell survival towards heat stress. The positive effect of calcium partly explained the protective mechanism of some calcium-containing protectants. The reported approach, which improves the stress tolerance of cells, can be combined with the optimization of spray drying conditions and protectant formulation for developing a consolidated powder production scheme.

Inactivation of cysteine 674 in the SERCA2 accelerates experimental aortic aneurysm

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis. SERCA2 cysteine 674 (C674) is highly conservative and its irreversible oxidation is upregulated in human and mouse aortic aneurysms, especially in smooth muscle cells (SMCs). The contribution of SERCA2 and its redox C674 in the development of aortic aneurysm remains enigmatic. Objective: Our goal was to investigate the contribution of inactivation of C674 to the development of aortic aneurysm and the mechanisms involved. Approach and results: Using SERCA2 C674S knock-in (SKI) mouse line, in which half of C674 was substituted by serine 674 (S674) to represent partial irreversible oxidation of C674 in aortic aneurysm, we found that in aortic SMCs the replacement of C674 by S674 resulted in SMC phenotypic modulation. In SKI SMCs, the increased intracellular calcium activated calcium-dependent calcineurin, which promoted the nuclear translocation of nuclear factor of activated T-lymphocytes (NFAT) and nuclear factor kappa-B (NFκB), while inhibition of calcineurin blocked SMC phenotypic modulation. Besides, the replacement of C674 by S674 accelerated angiotensin II-induced aortic aneurysm. Conclusions: Our results indicate that the inactivation of C674 by causing the accumulation of intracellular calcium to activate calcineurin-mediated NFAT/NFκB pathways, resulted in SMC phenotypic modulation to accelerate aortic aneurysm, which highlights the importance of C674 redox state in the development of aortic aneurysms.

Calcium signaling in brain microvascular endothelial cells and its roles in the function of the blood-brain barrier.

The blood-brain barrier (BBB) plays critical roles in maintaining the stability of the brain's internal milieu, providing nutrients for the brain, and preventing toxic materials from the blood from entering the brain. The cellular structure of the BBB is mainly composed of brain microvascular endothelial cells (BMVECs), which are surrounded by astrocytic endfeet that are connected by tight junction proteins, pericytes and astrocytes. Recently, several studies have shown that aberrant increase in intracellular calcium levels in BMVECs lead to cellular metabolic disturbances and subsequent impairment of BBB integrity. Although multiple stresses can lead to intracellular calcium accumulation, inherent protective mechanisms in affected cells are subsequently activated to maintain calcium homeostasis. However, once the increase in intracellular calcium goes beyond a certain threshold, disturbances in cellular structures, protein expression, and the BBB permeability are inevitable. Here, we review recent research on the different factors regulating intracellular calcium concentrations and the mechanisms related to how calcium signaling cascades protect the BMVECs from outside injury. We also consider the potential of calcium signaling regulators as therapeutic targets for modulating intracellular calcium homeostasis and ameliorating BBB disruption in patients with calcium-related pathologies.

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The search for new more effective techniques to repair bone fractures and defects is an urgent task of healthcare. Objective To explore the efficacy of a preparation containing etidronates of lanthanide and calcium ions in regenerative repair of bone defects. Material and methods The osteoblastic MC3T3-E1 and the osteoclastic RAW 264.7 cell lines were used in in vitro experiments at the first stage of the research. The agent was postoperatively injected in a bone defect of 36 rabbits on days 3 and 5 to assess the preparation’s effect on regenerative repair of small defects with diameter of 2.5 mm. Radiometric and reactive morphological characteristics of bone tissue were evaluated at the fracture site at the beginning, middle and end of experiment. Results The above preparation was found to enhance osteogenic differentiation and stimulate accumulation of intracellular calcium in MC3T3 E1 cells. However, the preparation was not shown to inhibit RANKL-induced osteoclast differentiation in vitro. Histological and computed tomography findings demonstrated statistically significant differences between control and experimental animals (p less 0.01) and indicated to the preparation’s effect of promoting regenerative repair of small bone defects. Conclusion The series was the first to show the effect of the preparation containing etidronates of lanthanide and calcium ions as stimulating osteoblast activity in vitro and promoting early regenerative repair of small bone defects.

Prokineticin 2 overexpression induces spermatocyte apoptosis in varicocele in rats.

Varicocele is one of the most important causes of male infertility, as this condition leads to a decline in sperm quality. It is generally believed that the presence of varicocele induces an increase in reactive oxygen species levels, leading to oxidative stress and sperm apoptosis; however, the specific pathogenic mechanisms affecting spermatogenesis remain elusive. Prokineticin 2 (PK2), a secretory protein, is associated with multiple biological processes, including cell migration, proliferation, and apoptosis. In the testis, PK2 is expressed in spermatocytes under normal physiological conditions. To investigate the role of PK2 in varicocele, a rat varicocele model was established to locate and quantify the expression of PK2 and its receptor, prokineticin receptor 1 (PKR1), by immunohistochemistry and quantitative real-time PCR assays (qPCR). Moreover, H2O2 was applied to mimic the oxidative stress state of varicocele through coculturing with a spermatocyte-derived cell line (GC-2) in vitro, and the apoptosis rate was detected by flow cytometry. Here, we illustrated that the expression levels of PK2 and PKR1 were upregulated in the spermatocytes of the rat model. Administration of H2O2 stimulated the overexpression of PK2 in GC-2. Transfection of recombinant pCMV-HA-PK2 into GC-2 cells promoted apoptosis by upregulating cleaved-caspase-3, caspase-8, and B cell lymphoma 2-associated X; downregulating B cell lymphoma 2; and promoting the accumulation of intracellular calcium. Overall, we revealed that the varicocele-induced oxidative stress stimulated the overexpression of PK2, leading to apoptosis of spermatocytes. Our study provides new insight into the mechanisms underlying oxidative stress-associated male infertility and suggests a novel therapeutic target for male infertility.

Resveratrol pretreatment alleviates myocardial ischemia/reperfusion injury by inhibiting STIM1-mediated intracellular calcium accumulation.

Previous studies have shown that stromal interaction molecule1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation in H9C2 cells subjected to hypoxia/reoxygenation(H/R) injury. The aim of the present study was to investigate the effect of resveratrol on STIM1-mediated intracellular Ca2+ accumulation and subsequent cell death in the context of myocardial ischemia/reperfusion (I/R) injury. C57 BL/6 mice were fed with either saline or resveratrol (50mg/kg daily for 2weeks) and then subjected to myocardial I/R injury. TTC/Evans Blue staining and TUNEL assay were performed to quantify the infarct size and apoptosis index. The cardiac function was evaluated by echocardiography. Neonatal rat ventricular cardiomyocytes (NRVCs) underwent hypoxia/reoxygenation (H/R) to establish the in vitro model. To achieve over-expression, NRVCs were transfected with STIM1-adenovirus vector. Apoptosis was analyzed by TUNEL assay. Cell viability was measured using MTS assay and cell necrosis was determined by LDH release assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using a Fluo-3AM probe. Resveratrol significantly reduced apoptosis, decreased infarct size, and improved cardiac function in mice subjected to myocardial I/R injury. In NRVCs, resveratrol also downregulated STIM1 expression accompanied by decreased intracellular Ca2+ accumulation elicited by H/R injury. In addition, resveratrol reduced cell apoptosis, upregulated the Bcl-2, decreased Bax, and cleaved caspase-3 expression. Furthermore, the effects of resveratrol on STIM1-mediated intracellular Ca2+ accumulation, apoptotic proteins, and H/R-induced cell injury were exacerbated by STIM1 over-expression and were partly abolished by SOCE inhibitor SKF96365 in NRVCs in vitro. Our findings demonstrate that resveratrol exerts anti-apoptotic activity and improves cardiac functional recovery following myocardial I/R by inhibiting STIM1-induced intracellular Ca2+ accumulation.

Toll-Like Receptor-Mediated Activation of CD39 Internalization in BMDCs Leads to Extracellular ATP Accumulation and Facilitates P2X7 Receptor Activation.

Toll-like receptors (TLRs) trigger innate immune responses through their recognition of conserved molecular ligands of either endogenous or microbial origin. Although activation, function, and signaling pathways of TLRs were already well-studied, their precise function in specific cell types, especially innate immune cells, needs to be further clarified. In this study, we showed that when significantly decreased amounts of membrane CD39, an adenosine triphosphate (ATP)-degrading enzyme, were detected in lipopolysaccharide (LPS)-treated bone marrow-derived dendritic cells (BMDCs), Cd39 mRNA expression, and whole-cell CD39 expression were at the same levels as those in untreated BMDCs. Further experiments demonstrated that the downregulation of membrane CD39 expression in LPS-treated BMDCs was mediated by endocytosis, leading to membrane-exposed CD39 downregulation, which was positively associated with decreased enzymatic activity in ATP metabolism and increased extracellular ATP accumulation. The accumulated ATP promoted intracellular calcium accumulation and IL-1β production in BMDCs through P2X7 signaling activation. Further research revealed that not only LPS but also other TLR ligands, excluding polyI:C, induced CD39 internalization in BMDCs and that the MyD88 pathway was critical in this process. The results suggested that the activation of CD39 internalization in DCs induced by a TLR ligand caused increased ATP accumulation, leading to P2X7 receptor activation that mediated a proinflammatory effect. Considering the strong modulatory effect of extracellular ATP accumulation on the immune response and inflammation, the manipulation of membrane CD39 expression on DCs may have implications on the regulation and treatment of inflammatory responses.

Activation of CaMKKβ-AMPK-mTOR pathway is required for autophagy induction by β,β-dimethylacrylshikonin against lung adenocarcinoma cells

β,β-Dimethylacrylshikonin (DMAS), an active ingredient of Lithospermum erythrorhizon and Arnebia euchroma, possess anti-neoplasm properties. Recently, DMAS was reported to stimulate autophagy in lung adenocarcinoma cells. However, the mechanisms by which DMAS modulates autophagy. have not yet been clearly elucidated. In this study, we found that DMAS significantly elevated intracellular free calcium accumulation. This activated the CaMKKβ-AMPK-mTOR pathway, subsequently inhibited mTOR and its substrate p70s6k and 4E-BP1, eventually leading to autophagy. In addition, we demonstrated that inhibition of autophagy by BAPTA-AM or STO-609 or compound C potently enhanced DMAS-induced lung adenocarcinoma cells apoptosis and growth inhibition. Overall, our results suggested that cytoprotective autophagy was triggered by DMAS via CaMKKβ-AMPK-mTOR signaling cascade in human lung adenocarcinoma cells, meaning that combining use of DMAS and autophagy inhibitors as a novel therapeutic option for lung adenocarcinoma will be very promising.

Modified age-dependent expression of NaV1.6 in an ALS model correlates with motor cortex excitability alterations

Cortical hyperexcitability is an early and intrinsic feature of Amyotrophic Lateral Sclerosis (ALS), but the mechanisms underlying this critical neuronal dysfunction are poorly understood. Recently, we have demonstrated that layer V pyramidal neurons (PNs) in the primary motor cortex (M1) of one-month old (P30) G93A ALS mice display an early hyperexcitability status compared to Control mice. In order to investigate the time-dependent evolution of the cortical excitability in the G93A ALS model, here we have performed an electrophysiological and immunohistochemical study at three different mouse ages. M1 PNs from 14-days old (P14) G93A mice have shown no excitability alterations, while M1 PNs from 3-months old (P90) G93A mice have shown a hypoexcitability status, compared to Control mice. These age-dependent cortical excitability dysfunctions correlate with a similar time-dependent trend of the persistent sodium current (INaP) amplitude alterations, suggesting that INaP may play a crucial role in the G93A cortical excitability aberrations. Specifically, immunohistochemistry experiments have indicated that the expression level of the NaV1.6 channel, one of the voltage-gated Na+ channels mainly distributed within the central nervous system, varies in G93A primary motor cortex during disease progression, according to the excitability and INaP alterations, but not in other cortical areas. Microfluorometry experiments, combined with electrophysiological recordings, have verified that P30 G93A PNs hyperexcitability is associated to a greater accumulation of intracellular calcium ([Ca2+]i) compared to Control PNs, and that this difference is still present when G93A and Control PNs fire action potentials at the same frequency.These results suggest that [Ca2+]i de-regulation in G93A PNs may contribute to neuronal demise and that the NaV1.6 channels could be a potential therapeutic target to ameliorate ALS disease progression.

Acupuncture attenuates cognitive impairment, oxidative stress and NF-κB activation in cerebral multi-infarct rats.

Patients with multiple infarct dementia (MID) have subtle deficits that commonly go unnoticed, and are at risk of developing Alzheimer's disease. Oxidative stress induced by ischaemic injury results in intracellular calcium accumulation and neuronal apoptosis, leading to cognitive impairment by triggering various cellular signal transduction pathways. Several studies have suggested that NF-κB in the presence of p53 has a pro-apoptotic function in various models, but the mechanism is unclear. The aim of this study was to investigate whether acupuncture could protect cognitive function against cerebral multi-infarction (CMi) induced oxidative stress by inhibiting the activation of NF-κB and its target gene p53. An animal model of CMi was established by injecting homologous blood emboli into the right internal carotid artery of male Wistar rats. After 2 weeks of acupuncture treatment, cognitive function was detected by novel object recognition. Electron spin resonance and Fluo-3 fuorescence imaging were used to test the generation of ROS and intracellular calcium accumulation, respectively. Expression of NF-κB and p53 was examined by Western blot analysis and immunofluorescence. CMi induced spatial learning and memory impairment, overproduction of intracellular hydroxyl radicals, and elevations of Ca2+, which were ameliorated by verum acupuncture treatment. Acupuncture inhibited activation of NF-κB and its downstream target gene p53. These findings suggest that acupuncture could protect cognitive function against oxidative stress induced by CMi, which is partially associated with suppression of NF-κB-p53 activation.

Anti‐edematous effect of neurolysin in ischemic stroke

During ischemic stroke brain injury occurs due to oxygen and nutrient deficiency, which initiates secondary injury cascades, including intracellular sodium and calcium accumulation, release of neurotoxic dopamine and glutamate, ion gradient dissipation, accumulation of reactive oxygen species, mitochondrial dysfunction, and excitotoxicity. All of these pathophysiologic processes contribute to cellular and vasogenic edema, which can be a life‐threatening effect of stroke and the primary reason for patient mortality after stroke.Neuropeptides similar to neurotransmitters play a significant role in different signaling pathways. They act on the cellular surface after releasing into the extracellular spaces and their functions are terminated by endogenous neuropeptidases. Neurolysin (Nln), a zinc metallopeptidase, has been reported to be up‐regulated during stroke and plays a crucial role in the improvement of stroke outcomes. Functional importance of this enzyme in post‐stroke brain repair is due to its capability in processing a diverse group of neuropeptides, such as substance P, bradykinin and neurotensin, which all have distinct functions in the development of inflammation, excitotoxicity, and cell death after stroke. The objective of our present study was to investigate the role of Nln through the deactivation of the above neuropeptide substrates and subsequent reduction of brain edema after a model of ischemic stroke.For an ex‐vivo model of ischemia, an acute brain slice model has been used. Briefly, 5–7 month old CD1 male mouse brain was extracted and 400 μm slices were exposed to 1 hour of oxygen glucose deprivation (OGD) condition followed by reperfusion (R) for 2 hours in the presence and absence of Nln substrates, i.e., substance P, bradykinin and neurotensin. To study the role of Nln, Nln substrates were co‐incubated with Nln during OGD/R. We observed that the 21% water content in slices increased when exposed to OGD/R compared to normoxia which further increased in the presence of Nln substrates during reperfusion, suggesting the role of Nln substrate in edema formation. Interestingly, the presence of rNln attenuated the enhanced edema formation by Nln substrates. These results indicated the role of Nln in reducing brain edema during ischemic stroke. Furthermore, to confirm that the effect of Nln is through deactivation of neuropeptides, we confirmed the hydrolysis of neuropeptides in the presence of Nln by using the LC‐MS/MS method.These results imply that cellular edema formation after stroke can be worsened by common Nln substrates (substance P, bradykinin, and neurotensin), which all have well‐documented neurovascular effects that exacerbate brain edema formation, and treatment with rNln can significantly attenuate cerebral swelling. This study is instrumental in explaining the possible role of Nln substrates and their deleterious effect in edema formation during a stroke and further studies are needed to study the positive role of Nln activation in stroke recovery and reduction of brain edema.Support or Funding InformationThis research was supported by NIH R01 NS106879 to VK and TA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low doses of Bisphenol A have pro-inflammatory and pro-oxidant effects, stimulate lipid peroxidation and increase the cardiotoxicity of Doxorubicin in cardiomyoblasts

Endocrine disrupters are strictly associated to cancer and several cardiovascular risk factors. Bisphenol A (BPA) is an endocrine disrupter commonly used in the manufacturing of plastics based on polycarbonate, polyvinyl chloride and resins. Our study aims to investigate whether BPA may cause pro-oxidative and pro-inflammatory effects on cardiomyoblasts, thus exacerbating the Doxorubicin (DOXO)-induced cardiotoxicity phenomena. We tested the metabolic effects of BPA at low doses analyzing its affections on the intracellular calcium uptake, oxidative stress, lipid peroxidation and production of nitric oxide and interleukins. Co-incubation of BPA and DOXO significantly reduced the cardiomyoblast viability, compared to only DOXO exposure cells. The mechanisms underlying these effects are based on the stimulation of the intracellular calcium accumulation and lipid peroxidation. Notably, BPA increase the production of pro-inflammatory interleukins involved in cardiovascular diseases as well as in DOXO-Induced cardiotoxicity phenomena. This study provides a rationale for translational studies in the field of cardio-oncology.

MiR‑223‑3p/TIAL1 interaction is involved in the mechanisms associated with the neuroprotective effects of dexmedetomidine on hippocampal neuronal cells invitro.

Dexmedetomidine (DEX), an α2 adrenoceptor agonist, is a commonly used anesthetic drug in surgical procedures. Previous studies have indicated that DEX exerts neuroprotective effects. However, the molecular mechanism underlying this process remains to be elucidated. The present study investigated a potential implication of microRNA (miR)-223-3p in the DEX-induced anti-oxidative effect on neuronal cells. The results indicated that following hydrogen peroxide (H2O2)-mediated induction of oxidative stress, the viability of human hippocampal neuronal cells was markedly decreased, as determined by an MTT assay. In addition, treatment with H2O2 induced cell apoptosis, the release of lactate dehydrogenase, accumulation of intracellular calcium, phosphorylation of calmodulin-2, and production of malondialdehyde and reactive oxygen species. Furthermore, treatment with H2O2 inhibited the expression of mir-223-3p and enhanced the expression of its target cytotoxic granule associated RNA binding protein like 1 (TIAL1), and these effects were reversed by treatment with DEX. Mechanistic studies demonstrated that the 3′-untranslated region of TIAL1 is a direct target of mir-223-3p. The results of the present study demonstrated that DEX may induce its neuroprotective effects by regulating the interaction between miR-223-3p and TIAL1. Therefore, the manipulation of miR-223-3p/TIAL1 interaction may be involved in the neuroprotective effects of DEX.

Microglial Calcium Release-Activated Calcium Channel Inhibition Improves Outcome from Experimental Traumatic Brain Injury and Microglia-Induced Neuronal Death.

Store-operated Ca2+ entry (SOCE) mediated by calcium release-activated calcium (CRAC) channels contributes to calcium signaling. The resulting intracellular calcium increases activate calcineurin, which in turn activates immune transcription factor nuclear factor of activated T cells (NFAT). Microglia contain CRAC channels, but little is known whether these channels play a role in acute brain insults. We studied a novel CRAC channel inhibitor to explore the therapeutic potential of this compound in microglia-mediated injury. Cultured microglial BV2 cells were activated by Toll-like receptor agonists or IFNγ. Some cultures were treated with a novel CRAC channel inhibitor (CM-EX-137). Western blots revealed the presence of CRAC channel proteins STIM1 and Orai1 in BV2 cells. CM-EX-137 decreased nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression in activated microglia and reduced agonist-induced intracellular calcium accumulation in microglia, while suppressing inflammatory transcription factors nuclear factor kappa B (NF-κB) and nuclear factor of activated T cells (NFAT). Male C57/BL6 mice exposed to experimental brain trauma and treated with CM-EX-137 had decreased lesion size, brain hemorrhage, and improved neurological deficits with decreased microglial activation, iNOS and Orai1 and STIM1 levels. We suggest a novel anti-inflammatory approach for managing acute brain injury. Our observations also shed light on new calcium signaling pathways not described previously in brain injury models.

Second signals rescue B cells from activation-induced mitochondrial dysfunction and death.

B cells are activated by two temporally distinct signals, the first provided by antigen binding to the B cell antigen receptor (BCR) and the second by T helper cells. Here we show that B cells responded to antigen by rapidly increasing metabolic activity including both oxidative phosphorylation and glycolysis. In the absence of a second signal B cells progressively lost mitochondrial function and glycolytic capacity leading to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response activated calcium channels that was preventable for approximately nine hours after B cell antigen binding by either T helper cells or Toll-like receptor 9 signaling. Thus, BCR signaling appears to activate a metabolic program that imposes a limited time window in which B cells either receive a second signal and survive or are eliminated.

Real-Time Signaling Assays Demonstrate Somatostatin Agonist Bias for Ion Channel Regulation in Somatotroph Tumor Cells.

Acromegaly is a neuroendocrine disorder caused by excess secretion of GH by somatotroph tumor cells. It is often treated with somatostatin receptor (SSTR) 2 agonists, which suppress GH secretion. SOM230 is a somatostatin analogue that targets multiple SSTRs and was recently approved for patients with treatment-resistant acromegaly. Previous reports indicate that SOM230 may function as a biased agonist, suggesting that its ability to selectively activate SSTR-dependent signaling events may contribute to its therapeutic efficacy. To better understand how SOM230 modulates Sstr2A function, which is the most commonly expressed SSTR in somatotrophs, we used real-time assays to study SOM230-dependent signaling in rat pituitary tumor cells. We observed that SOM230 suppressed cAMP production in a Gαi-dependent manner, similar to conventional Sstr2A agonists. However, it did not cause receptor internalization as would be expected for an Sstr2A agonist. Surprisingly, SOM230 did not cause membrane hyperpolarization, which is an important mechanism by which Sstr2a activation suppresses intracellular calcium (Ca2+) accumulation and GH secretion. In fact, SOM230 inhibited the ability of conventional somatostatin analogues to control membrane potential. However, SOM230 still inhibited intracellular Ca2+ accumulation in a novel, Gβγ-dependent manner. These studies show that SOM230 exhibits strong agonist bias in regulating signaling pathways downstream of Sstr2A that control GH secretion.

Tacrolimus-Induced Apoptosis is Mediated by Endoplasmic Reticulum–derived Calcium-dependent Caspases-3,-12 in Jurkat Cells

Apoptotic signal pathways are delivered to caspase-3, caspase-9, or both in different cells via the death receptor pathway, mitochondrial pathway, or by the endoplasmic reticulum (ER) pathway through initiators of caspase-3, -8, -9, or -12. Tacrolimus (Tac)–induced apoptosis was characterized by nuclear fragmentation and caspase-3 activation. We examined the effect of tacrolimus on ER-derived calcium and caspase-3,-12–mediated apoptosis on Jurkat human T lymphocyte. Tac decreased the viability of Jurkat cells in a dose-dependent manner. Tac also increased continuously intracellular concentration of calcium from 24 hours to 72 hours. We did not find intracellular calcium changes on the treatment of calcium ionorpore (A23187) regardless of 1 nmol/L Tac concentration level. However, calcium adenosine triphosphatase inhibitor (thapsigargin) increased intracellular calcium accumulation and co-treating 1 nmol/L Tac further induced intracellular calcium accumulation. Interestingly, we found that 1 nmol/L Tac treatment induced activation of caspase-12 protease as well as the catalytic activity of caspase-3 but not catalytic activation of caspase-6, -8, and -9 proteases in Jurkat cells. These data advance our understanding of Tac-induced apoptosis is ER-derived calcium and caspases-3,-12– mediated apoptosis in human Jurkat cell line.

Antigen binding to B cells activates a metabolic program that in the absence of a second signal leads to mitochondrial dysfunction and cell death

Abstract Activation of B cells to proliferate and differentiate into antibody secreting cells is regulated to avoid unwanted antibody responses by two temporally distinct signals, the first provided by antigen binding to the B cell receptor (BCR) and the second by T helper cells. Despite the central role of these two signals in driving antibody responses, our understanding of the consequences of each signal is incomplete. Here using in vitro and in vivo models, we investigated the metabolic changes that accompany B cell activation and show that antigen binding to the BCR signals for rapid increases in both oxidative phosphorylation and glycolysis, however early events following antigen engagement including spreading and contraction of antigen bound BCR and internalization of the bound antigen depend solely on the energy produced by oxidative phosphorylation. Changes in gene expression, mitochondrial performance and glucose uptake follow the initial activation, preparing the cells to respond to a second signal. However, in the absence of a second signal B cells undergo progressive loss of mitochondria function and glycolytic capacity ultimately leading to apoptosis. Mitochondria dysfunction is a result of the gradual accumulation of intracellular calcium which leads to inefficient oxidative phosphorylation and increased ROS production and eventually swollen patches of mitochondria. This process can be avoided for approximately nine hours after B cell antigen binding by either T helper cells or by signaling through Toll-like receptor 9. Thus, antigen binding to B cells appears to activate a metabolic program that imposes a short time window in which B cells either receive a second signal and survive or alternatively face elimination.