Endoplasmic Reticulum Calcium Dynamics Research Articles

Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.

Panax Notoginseng Saponins Protect Cardiac Myocytes Against Endoplasmic Reticulum Stress and Associated Apoptosis Through Mediation of Intracellular Calcium Homeostasis.

Endoplasmic reticulum (ER) stress has been demonstrated to play important roles in the pathogenesis of various cardiovascular diseases. The ER stress pathway is therefore a promising therapeutic target in cardiovascular disease. Although Panax notoginseng saponins (PNS) are one of the patent medicines that are traditionally used to treat cardiovascular disorders, their effects on ER stress in cardiac myocytes remain unexploited so far. This study investigates the effects of PNS on ER stress and its associated cell apoptosis along with the related mechanism in cardiac myocytes. PNS compounds were identified via high-performance liquid chromatograph (HPLC) assay. PNS-pretreated H9c2 cells, HL-1 cells, and primary cultured neonatal rat cardiomyocytes were stimulated with thapsigargin (TG) to induce ER stress response and apoptosis. ER stress response was tested by immunofluorescence or immunoblot of the ER protein chaperones—calnexin, binding immunoglobulin protein (BiP) and the C/EBP homologous protein (CHOP). Cell viability was tested by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was detected by immunoblot of Cleaved caspase-3 and flow cytometry analysis of Annexin V/propidium iodide (PI) staining. Cytosolic, mitochondrial, and ER calcium dynamics were investigated by calcium imaging. Moreover, a ryanodine receptor type-2 (RyR2) overexpression stable cell line was generated to verify the mechanism of RyR2 involved in PNS in the inhibition of ER stress and cell apoptosis. We demonstrate here that PNS protected cardiac myocytes from ER stress response and associated cell death in a concentration-dependent manner. Importantly, PNS reduced the elevation of cytosolic calcium, mitochondria calcium, as well as ER calcium in response to either TG or histamine treatment. PNS protection in ER stress was regulated by RyR2 expression. In summary, PNS protection against TG-induced ER stress response and its associated cell apoptosis in cardiac myocytes is calcium dependent. Through the regulation of ER calcium release mediated by RyR2, a novel mechanism for PNS in the prevention of cardiovascular diseases is thereby identified.

Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis.

Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA) (that act through the FFA receptors) on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of second messenger pathway interactions will improve understanding of critical regulatory sites, how different GPCRs interact and pharmacological targets for modulating insulin secretion in type 2 diabetes.

T-Type voltage-sensitive calcium channels mediate mechanically-induced intracellular calcium oscillations in osteocytes by regulating endoplasmic reticulum calcium dynamics

One of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular calcium (Ca2+), and osteocytes in particular exhibit robust oscillations in Ca2+ when subjected to loading. Previous studies implicate roles for both the endoplasmic reticulum (ER) and T-Type voltage-sensitive calcium channels (VSCC) in these responses, but their interactions or relative contributions have not been studied. By observing Ca2+ dynamics in the cytosol (Ca2+cyt) and the ER (Ca2+ER), the focus of this study was to explore the role of the ER and T-Type channels in Ca2+ signaling in bone cells. We demonstrate that inhibition of T-Type VSCC in osteocytes significantly reduces the number of Ca2+cyt responses and affects Ca2+ER depletion dynamics. Simultaneous observation of Ca2+ exchange among these spaces revealed high synchrony between rises in Ca2+cyt and depressions in Ca2+ER, and this synchrony was significantly reduced by challenging T-Type VSCC. We further confirmed that this effect was mediated directly through the ER and not through store-operated Ca2+ entry (SOCE) pathways. Taken together, our data suggests that T-Type VSCC facilitate the recovery of Ca2+ER in osteocytes to sustain mechanically-induced Ca2+ oscillations, uncovering a new mechanism underlying the behavior of osteocytes as mechanosensors.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a <em>Gaussia</em> Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics.

Disrupted-in-schizophrenia-1 (DISC1) has emerged as a convincing susceptibility gene for multiple mental disorders, but its mechanistic link to the pathogenesis of schizophrenia related psychiatric conditions is yet to be further understood. Here, we showed that DISC1 localizes to the outer surface of the endoplasmic reticulum (ER). EXOC1, a subunit of the exocyst complex, interacted with DISC1 and affected its recruitment to inositol-1,4,5-trisphosphate receptor 1 (IP3R1). Notably, knockdown of DISC1 and EXOC1 elicited an exaggerated ER calcium response upon stimulation of IP3R agonists. Similar abnormal ER calcium responses were observed in hippocampal neurons from DISC1-deficient mutant mice. Moreover, perturbation of ER calcium dynamics upon DISC1 knockdown was effectively reversed by treatment with antipsychotic drugs, such as clozapine and haloperidol. These results collectively indicate that DISC1 is a regulatory factor in ER calcium dynamics, linking a perturbed intracellular calcium signaling and schizophrenia pathogenesis.

Quantitative Analysis of Receptors and Second Messengers Interactions in Pancreatic Beta Cell

The response of insulin secretion in pancreatic beta cell to nutrient stimuli and hormonal modulators is coordinated by the different receptors, messengers and signaling networks. We present an updated computational model of second messenger interactions in pancreatic beta cell that incorporate modern data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCRs), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways. Model includes glucagon like peptide 1 receptor, gastric inhibitory polypeptide receptor and adrenoreceptor for cAMP pathway regulation, and the muscarinic acetylcholine receptor and the fatty acid receptor (GPR40) for phospholipase C regulation. The values of most of the model parameters were inferred from available experimental data. Our analysis of the dynamic data provides evidence for a pivotal role for Ca2+-dependent adenylyl cyclase activation in the effect of glucagon-like peptide 1 on pancreatic β-cells. The regulatory properties of various adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose and glucagon-like peptide 1 on insulin secretion. On other hand, the regulatory properties of phospholipase C isoforms determine interaction of glucose, acetylcholine and fatty acids (that act through the receptor GPR40). We test the hypothesis that activation of specific key beta-cell GPCRs can be in some cases stimulate but in other combinations inhibit glucose-stimulated insulin secretion. The regulation of messenger's pathway interactions may be important pharmacological targets for improving insulin secretion in type 2 diabetes.

Abstract LB-80: Role of ER calcium in Bcl-2 resistance to dosage and duration-related cytotoxic efficacy of Taxol

Abstract Background and Objectives: Calcium is one of the key regulators in apoptosis. Among various calcium sources, our previous study shows that the endoplasmic reticulum (ER), the main intracellular calcium storage site, provides a specific target for the chemotherapy agent Taxol and thus contributes to apoptosis. Although the expression of the anti-apoptotic protein Bcl-2 has been shown to confer resistance to Taxol, the exact mechanism of how it inhibits apoptosis and the role of ER calcium in this process remains unclear. This study investigated whether ER-associated calcium changes were generated and related to resistance by Bcl-2 to Taxol-induced apoptosis. Materials and methods: Fluo4-AM and Cameleon D1ER was used to monitor cytosolic and ER calcium dynamics by advanced time-lapsed imaging. Annexin V-FITC and propidium iodide were used to detect various phases of apoptosis induced by Taxol at different dosages from 10−12 to 10−5 M and at different durations from 5 min to 12 hours. Breast cancer cell lines MDA-MB-M468 and MDA-MB-231 were used and Bcl-2 transfectant cell lines were constructed by stable transfection. Results and Significance: Our previous data shows that Taxol (above 10−6 M) induces a rapid ER calcium release (peaking at 5 minutes) that promotes apoptosis. In this study, upon the expression of Bcl-2, basal ER calcium levels are decreased and Taxol-induced ER calcium release is inhibited, playing a major role in Bcl-2 resistance to Taxol. Depending upon dose and exposure time, long-term (hours) Taxol treatment partially overcame the Bcl-2 inhibition of ER calcium release by continually stimulating the ER calcium release, ultimately inducing significant, although smaller apoptosis in the presence of Bcl-2 expression. In addition to the effect on ER calcium, our data shows that the extracellular calcium pool is involved in Bcl-2 resistance through increased capacitative calcium entry to refill the ER calcium store, preventing ER calcium depletion. Taxol does not have a significant effect on the enhanced calcium influx from the extracellular medium. Taken together, out results indicate that the ER calcium store is the focus of the Taxol attack and plays a key role in Bcl-2 resistance to cytotoxic efficacy of Taxol in breast cancer cells. This finding provides insight to better understand the complex relationship between Bcl-2, calcium and Taxol towards improving breast cancer chemotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-80. doi:1538-7445.AM2012-LB-80

TRPC1, STIM1, and ORAI Influence Signal-Regulated Intracellular and Endoplasmic Reticulum Calcium Dynamics in Human Myometrial Cells1

To explore the relationship between signal-stimulated increases in intracellular calcium ([Ca(2+)](i)) and depletion and refilling of the endoplasmic reticulum (ER) Ca(2+) stores ([Ca(2+)](L)) in human myometrial cells, we measured simultaneous changes in [Ca(2+)](i) and [Ca(2+)](L) using Fura-2 and Mag-fluo-4, respectively, in PHM1-41 immortalized and primary cells derived from pregnant myometrium and in primary cells derived from nonpregnant tissue. Signal- and extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and ER refilling stimulated by oxytocin and cyclopiazonic acid were not inhibited by voltage-operated channel blocker nifedipine or mibefradil, inhibition of Na(+)/Ca(2+) exchange with KB-R7943, or zero extracellular Na(+) in PHM1-41 cells. Gadolinium-inhibited oxytocin- and cyclopiazonic acid-induced SRCE and slowed ER store refilling. TRPC1 mRNA knockdown specifically inhibited oxytocin-stimulated SRCE but had no statistically significant effect on ER store refilling and no effect on either parameter following cyclopiazonic acid treatment. Dominant negative STIMΔERM expression attenuated oxytocin- and thapsigargin-stimulated SRCE. Both STIM1 and ORAI1-ORAI3 mRNA knockdowns significantly attenuated oxytocin- and cyclopiazonic acid-stimulated SRCE. The data also suggest that reduction in STIM1 or ORAI1-ORAI3 mRNA can impede the rate of ER store refilling following removal of SERCA inhibition. These data provide evidence for both distinct and overlapping influences of TRPC1, STIM1, and ORAI1-ORAI3 on SRCE and ER store refilling in human myometrial cells that may contribute to the regulation of myometrial Ca(2+) dynamics. These findings have important implications for understanding the control of myometrial Ca(2+) dynamics in relation to myometrial contractile function.

Taxol Directly Induces Endoplasmic Reticulum-Associated Calcium Changes That Promote Apoptosis in Breast Cancer Cells

Calcium, a key regulator of cell survival, is also important in regulating apoptosis. Although the chemotherapeutic agent Taxol employs apoptosis to induce cell death, the exact mechanism of how it induces apoptosis and the role of calcium in this process remains unclear. The main intracellular calcium storehouse, the endoplasmic reticulum, was identified as a new important gateway in apoptosis, possibly providing a target for Taxol. The goal of this study was to investigate whether calcium changes associated with the endoplasmic reticulum, were directly or indirectly generated by Taxol at clinically relevant doses, and related to Taxol-induced apoptosis in breast cancer cells. Time-lapsed imaging techniques followed by an endoplasmic reticulum-targeted construct, cameleon D1ER, were used to monitor cytosol--endoplasmic reticulum calcium dynamics in MDA-MB-468 (Bcl-2 negative) and MCF 7 (Bcl-2 positive) breast carcinoma cells. Apoptosis levels were measured with Annexin V and Propidium Iodide (PI) using flow cytometry. In both cell lines, Taxol at 2.5μM (∼10(-6) M) was observed to induce significant internal calcium changes, first a rapid endoplasmic reticulum calcium release and a transient cytosolic calcium increase upon Taxol addition. After several hours of Taxol treatment, the endoplasmic reticulum calcium store was gradually depleted, and a sustained cytosolic calcium elevation was observed before significant induction of apoptosis. Inhibition of these calcium changes decreased Taxol-induced apoptosis levels. In contrast, 0.2μM Taxol (∼10(-7)M) induced only a slight cellular calcium change, not enough to regulate apoptosis. Our findings demonstrate that endoplasmic reticulum calcium stores provide a direct target for Taxol action and are important for induction of apoptosis, independent of Bcl-2 status. Furthermore, our results show for the first time, that the role of calcium in Taxol-induced endoplasmic reticulum-mediated apoptosis is dependent on Taxol dosage.

Manganese suppresses ATP-dependent intercellular calcium waves in astrocyte networks through alteration of mitochondrial and endoplasmic reticulum calcium dynamics

The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca 2+) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca 2+ dynamics in astrocytes, thereby suppressing Ca 2+ wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca 2+-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca 2+ wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca 2+ was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca 2+ dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1–10 μM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca 2+ waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca 2+ uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca 2+ levels in cells using the sarco/endoplasmic reticulum Ca 2+-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca 2+ at concentrations as low as 1 μM. Examination of ATP-stimulated changes in mitochondrial Ca 2+ indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca 2+. It is concluded that exposure of astrocytes to low concentrations of Mn 2+ results in sequestration of Ca 2+ within the mitochondria that reduces the available pool of releasable Ca 2+ within the ER, thereby inhibiting calcium wave activity.