Intracellular Calcium Signaling Pathways Research Articles

Preventive effect of propolis on cognitive decline in Alzheimer’s disease model mice

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer’s disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in Neuro-2A cells; moreover, propolis-induced [Ca2+]i elevations were suppressed prior to 24-h pretreatment with amyloid-β. To reveal the effect of [Ca2+]i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300–1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model.

Action mechanism of snake venom l-amino acid oxidase and its double-edged sword effect on cancer treatment: Role of pannexin 1-mediated interleukin-6 expression

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2

BackgroundEarly embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive.MethodsTo identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression.ResultsWe showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the ‘Window of implantation’ markers in human endometrial epithelial cells.ConclusionsThese findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.Graphical

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) inhibitors: a novel approach in small molecule discovery

The calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) plays a key role in regulation of intracellular calcium levels and signaling pathways. It is involved in activation of downstream signaling pathways that regulate various cellular processes. Dysregulation of CAMKK2 activity has been linked to various diseases including cancer, suggesting that CAMKK2 inhibitors might be beneficial in oncological, metabolic and inflammatory indications. The most pressing issues in small molecule discovery are synthesis feasibility, novel chemical structure and desired biological characteristics. To circumvent this constraint, we employed ‘DrugspaceX’ for rapid lead identification, followed by repositioning seven FDA-approved drugs for CAMKK2 inhibition. Further, first-level transformation (Set1 analogues) was performed in ‘DrugspaceX’, followed by virtual screening. The t-SNE visualization revealed that the transformations surrounding Rucaparib, Treprostinil and Canagliflozin are more promising for developing CAMKK2 inhibitors. Second, using the top-ranked Set1 analogues, Set2 analogues were generated, and virtual screening revealed the top-ranked five analogues. Among the top five Set2 analogues, DE273038_5 had the lowest docking score of −11.034 kcal/mol and SA score of 2.59, retaining the essential interactions with Hotspot residues LYS194 and VAL270 across 250 ns simulation period. When compared to the other four compounds, the ligand effectiveness score was 0.409, and the number of rotatable penalties was only three. Further, DE273038_5 after two rounds of transformations was discovered to be novel and had not been previously described in other databases. These data suggest that the new candidate DE273038_5 is likely to have inhibitory activity at the CAMKK2 active site, implying potential therapeutic use. Communicated by Ramaswamy H. Sarma

Inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) is overexpressed in cholangiocarcinoma and its expression correlates with S100 calcium-binding protein A4 (S100A4).

Cholangiocarcinoma (CCA) is the second most malignant neoplasm in the liver that arises from the biliary tree. CCA is associated with a poor prognosis, and the key players involved in its pathogenesis are still not well understood. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), can mediate intracellular calcium (Ca2+) signaling pathways via inositol 1,4,5-trisphosphate (InsP3), activating inositol 1,4,5-trisphosphate receptors (ITPRs) and regulating tumor growth. ITPR isoform 3 (ITPR3) is the main intracellular Ca2+ release channel in cholangiocytes. The effects of intracellular Ca2+ are mediated by calcium-binding proteins such as Calmodulin and S100 calcium-binding protein A4 (S100A4). However, the clinicopathological and biological significance of EGFR, ITPR3 and S100A4 in CCA remains unclear. Thus, the present work investigates the immunoexpression of these three proteins in 59 CCAs from patients who underwent curative surgical treatment and correlates the data with clinicopathological features and survival. High ITPR3 expression was correlated with CA 19-9 levels, TNM stage and lymph node metastasis (N). Furthermore, ITPR3 expression was increased in distal CCA compared to control bile ducts and intrahepatic and perihilar CCAs. These observations were confirmed by proteomic analysis. ITPR3 and S100A4 clinical scores were significantly correlated. Furthermore, it was demonstrated that EGF induces calcium signaling in a cholangiocarcinoma cell line and ITPR3 colocalizes with nonmuscle myosin IIA (NMIIA). In summary, ITPR3 overexpression could contribute to CCA progression and it may represent a potential therapeutic target.

Plasmonic Imaging of Dynamic Interactions between Membrane Receptor Clusters beyond the Diffraction Limit in Live Cells.

As a general mechanism, ligand-induced receptor clustering on cell membrane plays determinative roles in pattern recognition and transmembrane signaling. Nevertheless, probing the dynamic characteristics for the complicated interactions between receptor clusters remains difficult because of the lack of strategy for receptor cluster labeling and long-term monitoring in live cells. Herein, we proposed a data-mining-integrated plasmon coupling microscopy to study the dynamic cluster-cluster interactions on cell surface. The receptor clusters were activated and labeled with multivalent plasmonic nanoprobes, which enables the real-time monitoring of individual receptor clusters and the measurement of cluster-cluster interactions from the analysis of plasmonic coupling for the nanoprobe pairs beyond the diffraction limit. Using this method, we found that the protease-activated receptor 1 (PAR1) clusters would experience an initial contact and then form a weakly bound cluster-cluster complex, followed by cluster fusion to generate large-sized signaling complexes. The underlying state transitions for the cluster-cluster fusion process were uncovered using a data-mining technique named the K-means-based hidden Markov model with the scattering intensity of coupled nanoprobe pairs as observations. All of the findings from single-particle analysis and bulk measurements suggested that the allosteric inhibitors could suppress the dynamic transitions from the weakly bound cluster-cluster complexes to fused signaling complexes, leading to the subsequent downregulation of intracellular calcium signaling pathways. We believe that this strategy is promising for imaging and monitoring receptor clustering as well as protein phase separation on the cell surface in various biological and physiological processes.

Antioxidant Properties of Hydrogen Gas Attenuates Oxidative Stress in Airway Epithelial Cells.

Oxidative stress plays a crucial role in the development of airway diseases. Recently, hydrogen (H2) gas has been explored for its antioxidant properties. This study investigated the role of H2 gas in oxidative stress-induced alveolar and bronchial airway injury, where A549 and NCI-H292 cells were stimulated with hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) in vitro. Results show that time-dependent administration of 2% H2 gas recovered the cells from oxidative stress. Various indicators including reactive oxygen species (ROS), nitric oxide (NO), antioxidant enzymes (catalase, glutathione peroxidase), intracellular calcium, and mitogen-activated protein kinase (MAPK) signaling pathway were examined to analyze the redox profile. The viability of A549 and NCI-H292 cells and the activity of antioxidant enzymes were reduced following induction by H2O2 and LPS but were later recovered using H2 gas. Additionally, the levels of oxidative stress markers, including ROS and NO, were elevated upon induction but were attenuated after treatment with H2 gas. Furthermore, H2 gas suppressed oxidative stress-induced MAPK activation and maintained calcium homeostasis. This study suggests that H2 gas can rescue airway epithelial cells from H2O2 and LPS-induced oxidative stress and may be a potential intervention for airway diseases.

The Effect of Cyclosporine A on Proteins Controlling Intracellular Calcium Concentration in Breast Cancer Cells.

Cyclosporine A (CsA) is an immunosuppressive drug commonly used to prevent autoimmune diseases. At the same time, CsA is a calcineurin (CaN) inhibitor. It affects the intracellular calcium signaling pathway. The effect of CsA on breast cancer cells, MDA-MB-231, plasma membrane calcium pump 1 (PMCA1), calmodulin (CaM), calcineurin (CaN), and cMyc, which are proteins that affect calcium signaling, were investigated. CsA inhibited the proliferation of MDA-MB-231 cells but did not affect the migration of the cells. After 24h of incubation, CsA suppressed the PMCA1 protein, which pumps intracellular calcium out of the cell. At the same time, calcium started to accumulate inside the cell and CaM protein was expressed, while PMCA1 was suppressed. The CaN protein was suppressed 72h after the administration of CsA, but the cMyc protein was expressed. Interestingly, 24h incubation when the PMCA1 protein is down-regulated after the duration of time, the cMyc protein is also down-regulated. Although the indirect effect of CaN and cMyc is known, this relationship between PMCA1 and cMyc was not known. As a result, it has been shown that CsA affects the PMCA pump by disrupting the intracellular calcium pathway in breast cancer cells.

Stress-derived reactive oxygen species enable hemocytes to release activator of growth blocking peptide (GBP) processing enzyme

Insect cytokine growth blocking peptide (GBP) is synthesized as an inactive precursor, termed proGBP, that is normally present in a significant concentration in the hemolymph of non-stressed animals (Hayakawa, 1990, 1991). Under stress conditions, proGBP is instantly processed to active GBP by a serine protease and this is thought to be an important initial step for insects to cope with stress-induced adverse effects via GBP-induced physiological changes. However, the detailed mechanism underlying proteolytic processing of hemolymph proGBP in insects under stress conditions remains unknown. Here we demonstrated that proGBP processing requires ROS-induced release of a proteinaceous factor from hemocytes that activates the inactive proGBP processing enzyme. The release of the activator protein from hemocytes is initiated by an elevation of the cytoplasmic Ca2+ concentration induced by ROS. Therefore, we concluded that stress-induced activation of proGBP requires ROS-dependent stimulation of an intracellular calcium signaling pathway in hemocytes, followed by release of the hemocyte proteinaceous factor that specifically activates the proGBP processing enzyme.

Thyroid hormones stimulate L-arginine transport in human endothelial cells

Thyroid hormone activity is associated with L-arginine metabolism and nitric oxide (NO) production, which participate in the cardiovascular manifestations of thyroid disorders. L-arginine transporters play an important role in activating L-arginine uptake and NO production. However, the effects of thyroid hormones on L-arginine transporters in endothelial cells have not yet been evaluated. The following methods were used. We measured L-arginine uptake, mRNA expression of L-arginine transporters, endothelial nitric oxide synthase (eNOS) mRNA and NO generation after the administration of T3, T4 and the T3 analog, 3,3′,5-triiodothyroacetic acid TRIAC in human umbilical vein endothelial cells (HUVECs). We also analyzed the role of αvβ3 integrin and of phosphatidyl-inositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPKs: ERK1/2, p38 and SAPK-JNK) and intracellular calcium signaling pathways as underlying mechanisms. To this end, αvβ3 integrin was pharmacologically inhibited by tetraiodothyroacetic acid (TETRAC) or genetically blocked by silencing αv mRNA and PI3K, MAPKs and intracellular calcium by selective inhibitors. The following results were obtained. Thyroid hormones and the T3 analog TRIAC increased L-arginine uptake in HUVECs, the sodium-independent y+/CAT isoforms, except CAT2b, sodium-dependent y+L system and sodium-independent system b0,+L-arginine transporters, eNOS mRNA and NO production. These effects were suppressed by αvβ3 integrin inhibition with TETRAC or αv integrin downregulation or by PI3K, MAPK or intracellular Ca2+ signaling inhibitors. In conclusion, we report for the first time that activation of L-arginine uptake by thyroid hormones is related to an upregulation of L-arginine transporters. This effect seems to be mediated by activation of αvβ3 integrin receptor and subsequent PI3K, MAPK and intracellular Ca2+ signaling pathways.

The Role of CCL18 protein in Breast Cancer Development and Progression

CCL18 is a CC chemokine ligand 18, which is produced by Tumor-Associated Macrophages (TAMs), that can stimulate the progressiveness, angiogenesis in breast cancer, as well as the underlying pathophysiological mechanisms and it was originally discovered as pulmonary and activation-regulated chemokine (PARC), dendritic cell (DC)-chemokine 1 (DC-CK1), alternative macrophage activation associated CC chemokine-1 (AMAC-1), and macrophage inflammatory protein-4 (MIP-4). The potential functional receptor of CCL18 protein is PITPNM3 that facilitates CCL18 effect and stimulates the intracellular calcium signaling pathway. Worldwide, Breast cancer (BCa) is one of the most common malignancies among the females in both developed and also developing county. This study mainly focused on the role of CCL18 in the production of breast tumor as well as in the invasiveness of breast cancer. The data here suggest that CCL18 protein play an important role in the development of breast tumor and also progression in breast cancer. This knowledge could be helpful for the proposal of new therapeutic approaches particularly in breast cancer

Parallel Stochastic Discrete Event Simulation of Calcium Dynamics in Neuron.

The intra-cellular calcium signaling pathways of a neuron depends on both biochemical reactions and diffusions. Some quasi-isolated compartments (e.g., spines) are so small and calcium concentrations are so low that one extra molecule diffusing in by chance can make a nontrivial difference in concentration (percentage-wise). These rare events can affect dynamics discretely in such a way that they cannot be evaluated by a deterministic and continuous simulation. Stochastic models of such a system provide a more detailed understanding of these systems than existing deterministic models because they capture their behavior at a molecular level. Our research focuses on the development of a high performance parallel discrete event simulation environment, Neuron Time Warp (NTW), which is intended for use in the parallel simulation of stochastic reaction-diffusion systems such as intra-calcium signaling. NTW is integrated with NEURON, a simulator which is widely used within the neuroscience community. We simulate two models, a calcium buffer and a calcium wave model. The calcium buffer model is employed in order to verify the correctness and performance of NTW by comparing it to a sequential deterministic simulation in NEURON. We also derived a discrete event calcium wave model from a deterministic model using the stochastic $\text{IP}_{3}\text{R}$IP3R structure.

Extra and intracellular calcium signaling pathway(s) differentially regulate histamine-induced myometrial contractions during early and mid-pregnancy stages in buffaloes (Bubalus bubalis)

This study examines the differential role of calcium signaling pathway(s) in histamine-induced uterotonic action during early and mid-pregnancy stages in buffaloes. Compared to mid pregnancy, tonic contraction, amplitude and mean-integral tension were significantly increased by histamine to produce myometrial contraction during early pregnancy with small effects on phasic contraction and frequency. Although uterotonic action of histamine during both stages of pregnancy is sensitive to nifedipine (a L-type Ca2+ channels blocker) and NNC55-0396 (T-type Ca2+ channels blocker), the role of extracellular calcium seems to be more significant during mid-pregnancy as in this stage histamine produced only 9.38±0.96% contraction in Ca2+ free-RLS compared to 21.60±1.45% in uteri of early pregnancy stage. Intracellular calcium plays major role in histamine-induced myometrial contraction during early pregnancy as compared to mid pregnancy, as in the presence of cyclopiazonic acid (CPA) Ca2+-free RLS, histamine produced significantly higher contraction in myometrial strips of early-pregancy in comparison to mid-pregnancy (10.59±1.58% and 3.13±0.46%, respectively). In the presence of U-73122, the DRC of histamine was significantly shifted towards right with decrease in maximal effect (Emax) only in early pregnancy suggesting the predominant role of phospholipase-C (PL-C) in this stage of pregnancy.

Dynamics of Calcium Signal and Leukotriene C4Release in Mast Cells Network Induced by Mechanical Stimuli and Modulated by Interstitial Fluid Flow

AbstractMast cells (MCs) play an important role in the immune system. Through connective tissues, mechanical stimuli activate intracellular calcium signaling pathways, induce a variety of mediators including leukotriene C4(LTC4) release, and affect MCs’ microenvironment. This paper focuses on MCs’ intracellular calcium dynamics and LTC4release responding to mechanical stimuli, explores signaling pathways in MCs and the effect of interstitial fluid flow on the transport of biological messengers and feedback in the MCs network. We use a mathematical model to show that (i) mechanical stimuli including shear stress induced by interstitial fluid flow can activate mechano-sensitive (MS) ion channels on MCs’ membrane and allow Ca2+entry, which increases intracellular Ca2+concentration and leads to LTC4release; (ii) LTC4in the extracellular space (ECS) acts on surface cysteinyl leukotriene receptors (LTC4R) on adjacent cells, leading to Ca2+influx through Ca2+release-activated Ca2+(CRAC) channels. An elevated intracellular Ca2+concentration further stimulates LTC4release and creates a positive feedback in the MCs network. The findings of this study may facilitate our understanding of the mechanotransduction process in MCs induced by mechanical stimuli, contribute to understanding of interstitial flow-related mechanobiology in MCs network, and provide a methodology for quantitatively analyzing physical treatment methods including acupuncture and massage in traditional Chinese medicine (TCM).

Nuclear factor of activated T-cell isoform expression and regulation in human myometrium.

BackgroundDuring pregnancy, myometrial gene and protein expression is tightly regulated to accommodate fetal growth, promote quiescence and ultimately prepare for the onset of labour. It is proposed that changes in calcium signalling, may contribute to regulating gene expression and that nuclear factor of activated T-cell (NFAT) transcription factors (isoforms c1-c4) may be involved. Currently, there is little information regarding NFAT expression and regulation in myometrium.MethodsThis study examined NFAT isoform mRNA expression in human myometrial tissue and cells from pregnant women using quantitative PCR. The effects of the Ca2+ ionophore A23187 and in vitro stretch (25 % elongation, static strain; Flexercell FX-4000 Tension System) on NFAT expression were determined in cultured human myometrial cells.ResultsHuman myometrial tissue and cultured cells expressed NFATc1-c4 mRNA. NFATc2 gene expression in cultured cells was increased in response to 6 h stretch (11.5 fold, P < 0.001, n = 6) and calcium ionophore (A23187, 5 μM) treatment (20.6 fold, P < 0.001, n = 6). This response to stretch was significantly reduced (90 %, P < 0.001, n = 10) in the presence of an intracellular calcium chelator, BAPTA-AM (20 μM).ConclusionsThese data suggest that NFATc2 expression is regulated by intracellular calcium and in vitro stretch, and that the stretch response in human myometrial cells is dependent upon intracellular calcium signalling pathways. Our findings indicate a potentially unique role for NFATc2 in mediating stretch-induced gene expression per se and warrant further exploration in relation to the mechanisms promoting uterine smooth muscle growth in early pregnancy and/or labour.

Searching for synergistic bronchodilators and novel therapeutic regimens for chronic lung diseases from a traditional Chinese medicine, Qingfei Xiaoyan Wan.

Classical Chinese pharmacopeias describe numerous excellent herbal formulations, and each prescription is an outstanding pool of effective compounds for drug discovery. Clarifying the bioactivity of the combined mechanisms of the ingredients in complex traditional Chinese medicine formulas is challenging. A classical formula known as Qingfei Xiaoyan Wan, used clinically as a treatment for prevalent chronic lung disease, was investigated in this work. A mutually enhanced bioactivity-guided ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) characterization system was proposed, coupled with a dual-luciferase reporter assay for β2AR-agonist cofactor screening. Arctiin, arctigenin, descurainoside and descurainolide B, four lignin compounds that showed synergistic bronchodilation effects with ephedrine, were revealed. The synergistic mechanism of arctigenin with the β2ARagonist involved with the reduction of free Ca2+ was clarified by a dual-luciferase reporter assay for intracellular calcium and the Ca2+ indicator fluo-4/AM to monitor changes in the fluorescence. The relaxant and contractile responses of airway smooth muscle are regulated by crosstalk between the intracellular cAMP and calcium signaling pathways. Our data indicated the non-selective βAR agonist ephedrine as the principal bronchodilator of the formula, whereas the lignin ingredients served as adjuvant ingredients. A greater understanding of the mechanisms governing the control of these pathways, based on conventional wisdom, could lead to the identification of novel therapeutic targets or new agents for the treatment of asthma and COPD.

Dissecting the molecular pathways of primary aldosteronism.

The great majority of the cases clinically diagnosed as primary aldosteronism (PA) have been caused by aldosterone-producing adenoma (APA) or idiopathic hyperaldosteronism (IHA). The differential diagnosis of both subtypes of PA is important due to the different therapeutic modes but clinically it is sometimes difficult. It is also important to understand the morphological features of these two subtypes with special emphasis upon differences of the status for aldosterone biosynthesis. In the last decade, molecular mechanisms of PA including the aberrant expression of G-protein coupled receptors (GPCRs), key regulators of the intracellular calcium signaling pathway and somatic mutations of ion channels, have been revealed and our understanding of the molecular pathways involved in excessive aldosterone production has been markedly advanced. In addition, newly developed monoclonal antibodies specific to the isoform of adrenal steroidogenic enzymes have demonstrated the novel profiles of adrenal steroidogenesis in PA. These novel findings indicate that the molecular mechanisms on the onset and pathophysiology of PA are more complicated than previously considered and further clarification of clinical relevance of these findings is required at this juncture.

Chronic blockade of extrasynaptic NMDA receptors ameliorates synaptic dysfunction and pro-death signaling in Huntington disease transgenic mice

In the YAC128 mouse model of Huntington disease (HD), elevated extrasynaptic NMDA receptor (Ex-NMDAR) expression contributes to the onset of striatal dysfunction and atrophy. A shift in the balance of synaptic–extrasynaptic NMDAR signaling and localization is paralleled by early stage dysregulation of intracellular calcium signaling pathways, including calpain and p38 MAPK activation, that couple to pro-death cascades. However, whether aberrant calcium signaling is a consequence of elevated Ex-NMDAR expression in HD is unknown. Here, we aimed to identify calcium-dependent pathways downstream of Ex-NMDARs in HD. Chronic (2-month) treatment of YAC128 and WT mice with memantine (1 and 10mg/kg/day), which at a low dose selectively blocks Ex-NMDARs, reduced striatal Ex-NMDAR expression and current in 4-month old YAC128 mice without altering synaptic NMDAR levels. In contrast, calpain activity was not affected by memantine treatment, and was elevated in untreated YAC128 mice at 1.5months but not 4months of age. In YAC128 mice, memantine at 1mg/kg/day rescued CREB shut-off, while both doses suppressed p38 MAPK activation to WT levels. Taken together, our results indicate that Ex-NMDAR activity perpetuates increased extrasynaptic NMDAR expression and drives dysregulated p38 MAPK and CREB signaling in YAC128 mice. Elucidation of the pathways downstream of Ex-NMDARs in HD could help provide novel therapeutic targets for this disease.

AMP‐ACTIVATED PROTEIN KINASE AS REGULATOR OF P2Y6 RECEPTOR‐INDUCED INSULIN SECRETION IN MOUSE PANCREATIC BETA CELLS

5′‐AMP‐activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular UDP activates P2Y6 receptors (P2Y6Rs) in pancreatic β cells to release insulin and reduce apoptosis, which would benefit diabetes. Here, we studied the role of P2Y6R in activation of AMPK and insulin secretion in MIN6 mouse pancreatic beta cells. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 activated AMPK, which was blocked by P2Y6R‐selective antagonist. Also, MRS2957 induced phosphorylation of acetyl‐coenzyme A carboxylase (ACC), a marker of AMPK activity. Calcium chelator BAPTA‐AM, calmodulin‐dependent protein kinase kinase (CaMKK) inhibitor STO‐069 and IP3 receptor antagonist 2‐APB attenuated P2Y6R‐mediated AMPK phosphorylation revealing involvement of intracellular calcium signaling pathways. P2Y6R agonist induced insulin secretion only at high glucose, which was reduced by AMPK gene silencing suggesting an involvement of AMPK in P2Y6 receptor‐mediated insulin secretion. Thus, P2Y6R has a crucial role in β cell function, suggesting its potential as a therapeutic target in diabetes.

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca2+ indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca2+ oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca2+ signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.