Calcium signalling silencing in atrial fibrillation.

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Greiser, Maura

2017-06-15

Subcellular calcium signalling silencing is a novel and distinct cellular and molecular adaptive response to rapid cardiac activation. Calcium signalling silencing develops during short-term sustained rapid atrial activation as seen clinically during paroxysmal atrial fibrillation (AF). It is the first 'anti-arrhythmic' adaptive response in the setting of AF and appears to counteract the maladaptive changes that lead to intracellular Ca 2+ signalling instability and Ca 2+ -based arrhythmogenicity. Calcium signalling silencing results in a failed propagation of the [Ca 2+ ] i signal to the myocyte centre both in patients with AF and in a rabbit model. This adaptive mechanism leads to a substantial reduction in the expression levels of calcium release channels (ryanodine receptors, RyR2) in the sarcoplasmic reticulum, and the frequency of Ca 2+ sparks and arrhythmogenic Ca 2+ waves remains low. Less Ca 2+ release per [Ca 2+ ] i transient, increased fast Ca 2+ buffering strength, shortened action potentials and reduced L-type Ca 2+ current contribute to a substantial reduction of intracellular [Na + ]. These features of Ca 2+ signalling silencing are distinct and in contrast to the changes attributed to Ca 2+ -based arrhythmogenicity. Some features of Ca 2+ signalling silencing prevail in human AF suggesting that the Ca 2+ signalling 'phenotype' in AF is a sum of Ca 2+ stabilizing (Ca 2+ signalling silencing) and Ca 2+ destabilizing (arrhythmogenic unstable Ca 2+ signalling) factors. Calcium signalling silencing is a part of the mechanisms that contribute to the natural progression of AF and may limit the role of Ca 2+ -based arrhythmogenicity after the onset of AF. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Calcium signal communication in the central nervous system.

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Braet, Katleen; Cabooter, Liesbet; Paemeleire, Koen; Leybaert, Luc

2004-02-01

The communication of calcium signals between cells is known to be operative between neurons where these signals integrate intimately with electrical and chemical signal communication at synapses. Recently, it has become clear that glial cells also exchange calcium signals between each other in cultures and in brain slices. This communication pathway has received utmost attention since it is known that astrocytic calcium signals can be induced by neuronal stimulation and can be communicated back to the neurons to modulate synaptic transmission. In addition to this, cells that are generally not considered as brain cells become progressively incorporated in the picture, as astrocytic calcium signals are reported to be communicated to endothelial cells of the vessel wall and can affect smooth muscle cell tone to influence the vessel diameter and thus blood flow. We review the available evidence for calcium signal communication in the central nervous system, taking into account a basic functional unit -the brain cell tripartite- consisting of neurons, glial cells and vascular cells and with emphasis on glial-vascular calcium signaling aspects.

Calcium as a signal integrator in developing epithelial tissues.

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Brodskiy, Pavel A; Zartman, Jeremiah J

2018-05-16

Decoding how tissue properties emerge across multiple spatial and temporal scales from the integration of local signals is a grand challenge in quantitative biology. For example, the collective behavior of epithelial cells is critical for shaping developing embryos. Understanding how epithelial cells interpret a diverse range of local signals to coordinate tissue-level processes requires a systems-level understanding of development. Integration of multiple signaling pathways that specify cell signaling information requires second messengers such as calcium ions. Increasingly, specific roles have been uncovered for calcium signaling throughout development. Calcium signaling regulates many processes including division, migration, death, and differentiation. However, the pleiotropic and ubiquitous nature of calcium signaling implies that many additional functions remain to be discovered. Here we review a selection of recent studies to highlight important insights into how multiple signals are transduced by calcium transients in developing epithelial tissues. Quantitative imaging and computational modeling have provided important insights into how calcium signaling integration occurs. Reverse-engineering the conserved features of signal integration mediated by calcium signaling will enable novel approaches in regenerative medicine and synthetic control of morphogenesis.

Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients

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Bading Hilmar

2007-07-01

Full Text Available Abstract Background In hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types. Results Using laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border. Conclusion Thus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.

Presynaptic calcium signalling in cerebellar mossy fibres

DEFF Research Database (Denmark)

Thomsen, Louiza Bohn; Jörntell, Henrik; Midtgaard, Jens

2010-01-01

Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX......)-sensitive fast Na(+) spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers....... Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon...

14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

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Michael P Grant

Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

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Bartlett, Paula J; Antony, Anil Noronha; Agarwal, Amit; Hilly, Mauricette; Prince, Victoria L; Combettes, Laurent; Hoek, Jan B; Gaspers, Lawrence D

2017-05-15

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca 2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca 2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca 2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) production and does not involve changes in the sensitivity of the IP 3 receptor or size of internal Ca 2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca 2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca 2+ ] i ) increases, which can adversely affect mitochondrial Ca 2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca 2+ -mobilizing hormones resulting in more sustained and prolonged [Ca 2+ ] i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca 2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) accumulation and phospholipase C

Extracellular signaling and multicellularity in Bacillus subtilis.

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Shank, Elizabeth Anne; Kolter, Roberto

2011-12-01

Bacillus subtilis regulates its ability to differentiate into distinct, co-existing cell types in response to extracellular signaling molecules produced either by itself, or present in its environment. The production of molecules by B. subtilis cells, as well as their response to these signals, is not uniform across the population. There is specificity and heterogeneity both within genetically identical populations as well as at the strain-level and species-level. This review will discuss how extracellular signaling compounds influence B. subtilis multicellularity with regard to matrix-producing cannibal differentiation, germination, and swarming behavior, as well as the specificity of the quorum-sensing peptides ComX and CSF. It will also highlight how imaging mass spectrometry can aid in identifying signaling compounds and contribute to our understanding of the functional relationship between such compounds and multicellular behavior. Copyright © 2011 Elsevier Ltd. All rights reserved.

Short-range intercellular calcium signaling in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas R

2005-01-01

The regulation of bone turnover is a complex and finely tuned process. Many factors regulate bone remodeling, including hormones, growth factors, cytokines etc. However, little is known about the signals coupling bone formation to bone resorption, and how mechanical forces are translated...... into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate...... whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally...

A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells.

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Long, Tingting; Su, Juan; Tang, Wen; Luo, Zhongling; Liu, Shuang; Liu, Zhaoqian; Zhou, Honghao; Qi, Min; Zeng, Weiqi; Zhang, Jianglin; Chen, Xiang

2013-10-01

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Requirement for nuclear calcium signaling in Drosophila long-term memory.

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Weislogel, Jan-Marek; Bengtson, C Peter; Müller, Michaela K; Hörtzsch, Jan N; Bujard, Martina; Schuster, Christoph M; Bading, Hilmar

2013-05-07

Calcium is used throughout evolution as an intracellular signal transducer. In the mammalian central nervous system, calcium mediates the dialogue between the synapse and the nucleus that is required for transcription-dependent persistent neuronal adaptations. A role for nuclear calcium signaling in similar processes in the invertebrate brain has yet to be investigated. Here, we show by in vivo calcium imaging of adult brain neurons of the fruit fly Drosophila melanogaster, that electrical foot shocks used in olfactory avoidance conditioning evoked transient increases in cytosolic and nuclear calcium concentrations in neurons. These calcium signals were detected in Kenyon cells of the flies' mushroom bodies, which are sites of learning and memory related to smell. Acute blockade of nuclear calcium signaling during conditioning selectively and reversibly abolished the formation of long-term olfactory avoidance memory, whereas short-term, middle-term, or anesthesia-resistant olfactory memory remained unaffected. Thus, nuclear calcium signaling is required in flies for the progression of memories from labile to transcription-dependent long-lasting forms. These results identify nuclear calcium as an evolutionarily conserved signal needed in both invertebrate and vertebrate brains for transcription-dependent memory consolidation.

The Hepatitis B Virus X Protein Elevates Cytosolic Calcium Signals by Modulating Mitochondrial Calcium Uptake

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Yang, Bei

2012-01-01

Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication. PMID:22031934

Short-range intercellular calcium signaling in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye

2005-01-01

into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate...... whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally...... different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other...

Extracellular matrix component signaling in cancer

DEFF Research Database (Denmark)

Multhaupt, Hinke A. B.; Leitinger, Birgit; Gullberg, Donald

2016-01-01

Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motil...... as well as matrix constitution and protein crosslinking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression. [on SciFinder(R)]...

Probabilistic encoding of stimulus strength in astrocyte global calcium signals.

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Croft, Wayne; Reusch, Katharina; Tilunaite, Agne; Russell, Noah A; Thul, Rüdiger; Bellamy, Tomas C

2016-04-01

Astrocyte calcium signals can range in size from subcellular microdomains to waves that spread through the whole cell (and into connected cells). The differential roles of such local or global calcium signaling are under intense investigation, but the mechanisms by which local signals evolve into global signals in astrocytes are not well understood, nor are the computational rules by which physiological stimuli are transduced into a global signal. To investigate these questions, we transiently applied receptor agonists linked to calcium signaling to primary cultures of cerebellar astrocytes. Astrocytes repetitively tested with the same stimulus responded with global signals intermittently, indicating that each stimulus had a defined probability for triggering a response. The response probability varied between agonists, increased with agonist concentration, and could be positively and negatively modulated by crosstalk with other signaling pathways. To better understand the processes determining the evolution of a global signal, we recorded subcellular calcium "puffs" throughout the whole cell during stimulation. The key requirement for puffs to trigger a global calcium wave following receptor activation appeared to be the synchronous release of calcium from three or more sites, rather than an increasing calcium load accumulating in the cytosol due to increased puff size, amplitude, or frequency. These results suggest that the concentration of transient stimuli will be encoded into a probability of generating a global calcium response, determined by the likelihood of synchronous release from multiple subcellular sites. © 2015 Wiley Periodicals, Inc.

Calcium and Nuclear Signaling in Prostate Cancer

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Ivan V. Maly

2018-04-01

Full Text Available Recently, there have been a number of developments in the fields of calcium and nuclear signaling that point to new avenues for a more effective diagnosis and treatment of prostate cancer. An example is the discovery of new classes of molecules involved in calcium-regulated nuclear import and nuclear calcium signaling, from the G protein-coupled receptor (GPCR and myosin families. This review surveys the new state of the calcium and nuclear signaling fields with the aim of identifying the unifying themes that hold out promise in the context of the problems presented by prostate cancer. Genomic perturbations, kinase cascades, developmental pathways, and channels and transporters are covered, with an emphasis on nuclear transport and functions. Special attention is paid to the molecular mechanisms behind prostate cancer progression to the malignant forms and the unfavorable response to anti-androgen treatment. The survey leads to some new hypotheses that connect heretofore disparate results and may present a translational interest.

High calcium concentration in bones promotes bone metastasis in renal cell carcinomas expressing calcium-sensing receptor.

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Joeckel, Elke; Haber, Tobias; Prawitt, Dirk; Junker, Kerstin; Hampel, Christian; Thüroff, Joachim W; Roos, Frederik C; Brenner, Walburgis

2014-02-28

The prognosis for renal cell carcinoma (RCC) is related to a high rate of metastasis, including 30% of bone metastasis. Characteristic for bone tissue is a high concentration of calcium ions. In this study, we show a promoting effect of an enhanced extracellular calcium concentration on mechanisms of bone metastasis via the calcium-sensing receptor (CaSR) and its downstream signaling molecules. Our analyses were performed using 33 (11/category) matched specimens of normal and tumor tissue and 9 (3/category) primary cells derived from RCC patients of the 3 categories: non-metastasized, metastasized into the lung and metastasized into bones during a five-year period after nephrectomy. Expression of CaSR was determined by RT-PCR, Western blot analyses and flow cytometry, respectively. Cells were treated by calcium and the CaSR inhibitor NPS 2143. Cell migration was measured in a Boyden chamber with calcium (10 μM) as chemotaxin and proliferation by BrdU incorporation. The activity of intracellular signaling mediators was quantified by a phospho-kinase array and Western blot. The expression of CaSR was highest in specimens and cells of patients with bone metastases. Calcium treatment induced an increased migration (19-fold) and proliferation (2.3-fold) exclusively in RCC cells from patients with bone metastases. The CaSR inhibitor NPS 2143 elucidated the role of CaSR on the calcium-dependent effects. After treatment with calcium, the activity of AKT, PLCγ-1, p38α and JNK was clearly enhanced and PTEN expression was almost completely abolished in bone metastasizing RCC cells. Our results indicate a promoting effect of extracellular calcium on cell migration and proliferation of bone metastasizing RCC cells via highly expressed CaSR and its downstream signaling pathways. Consequently, CaSR may be regarded as a new prognostic marker predicting RCC bone metastasis.

Extracellular nucleotide signaling in plants

Energy Technology Data Exchange (ETDEWEB)

Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

2016-09-08

Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility.

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Jian Sun

2016-05-01

Full Text Available A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling.

Sources of extracellular tau and its signaling.

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Avila, Jesús; Simón, Diana; Díaz-Hernández, Miguel; Pintor, Jesús; Hernández, Félix

2014-01-01

The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer's disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer's disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.

CBL-CIPK network for calcium signaling in higher plants

Science.gov (United States)

Luan, Sheng

Plants sense their environment by signaling mechanisms involving calcium. Calcium signals are encoded by a complex set of parameters and decoded by a large number of proteins including the more recently discovered CBL-CIPK network. The calcium-binding CBL proteins specifi-cally interact with a family of protein kinases CIPKs and regulate the activity and subcellular localization of these kinases, leading to the modification of kinase substrates. This represents a paradigm shift as compared to a calcium signaling mechanism from yeast and animals. One example of CBL-CIPK signaling pathways is the low-potassium response of Arabidopsis roots. When grown in low-K medium, plants develop stronger K-uptake capacity adapting to the low-K condition. Recent studies show that the increased K-uptake is caused by activation of a specific K-channel by the CBL-CIPK network. A working model for this regulatory pathway will be discussed in the context of calcium coding and decoding processes.

Differential effects of the steaming time and frequency for manufactured red Liriope platyphylla on nerve growth factor secretion ability, nerve growth factor receptor signaling pathway and regulation of calcium concentration.

Science.gov (United States)

Choi, Sun Il; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Hye Ryun; Lee, Young Ju; Son, Hong Joo; Lee, Hee Seob; Lee, Jong Sup; Kim, Hak Jin; Hwang, Dae Youn

2012-11-01

The herb Liriope platyphylla (LP) has been considered to have curative properties for diabetes, asthma and neurodegenerative disorders. To examine the effects of steaming time and frequency of manufactured red LP (RLP) on the nerve growth factor (NGF) secretion ability and NGF receptor signaling pathway, the NGF concentration, cell differentiation, NGF signaling pathway and calcium concentration were analyzed in neuronal cells treated with several types of LPs manufactured under different conditions. The maximum NGF secretion was observed in B35 cells treated with 50 µg/ml LP extract steamed for 9 h (9-SLP) and with two repeated steps (3 h steaming and 24 h air-dried) carried out 7 times (7-SALP). No significant changes in viability were detected in any of the cells treated with the various LPs, with the exception of 0-SLP and 0-SALP. In addition, PC12 cell differentiation was induced by treatment with the NGF-containing conditional medium (CM) collected from the RLP-treated cells. The levels of TrkA and extracellular signal-regulated kinase (ERK) phosphorylation in the high affinity NGF receptor signaling pathway were significantly higher in the cells treated with 3-SLP or 1-SALP/3-SALP CM compared with those treated with the vehicle CM. In the low affinity NGF receptor pathway, the expression levels of most components were higher in the 9-, 15- and 24-SALP CM-treated cells compared with the vehicle CM-treated cells. However, this level was significantly altered in cells treated with 3-SALP CM. Furthermore, an examination of the RLP function on calcium regulation revealed that only the LP- or RLP-treated cells exhibited changes in intracellular and extracellular calcium levels. RLP induced a significant decrease in the intracellular calcium levels and an increase in the extracellular calcium levels. These results suggest the possibility that steaming-processed LP may aid in the relief of neurodegenerative diseases through the NGF secretion ability and NGF

Calcium signals and caspase-12 participated in paraoxon-induced apoptosis in EL4 cells.

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Li, Lan; Cao, Zhiheng; Jia, Pengfei; Wang, Ziren

2010-04-01

In order to investigate whether calcium signals participate in paraoxon (POX)-induced apoptosis in EL4 cells, real-time laser scanning confocal microscopy (LSCM) was used to detect Ca(2+) changes during the POX application. Apoptotic rates of EL4 cells and caspase-12 expression were also evaluated. POX (1-10nM) increased intracellular calcium concentration ([Ca(2+)]i) in EL4 cells in a dose-dependent manner at early stage (0-2h) of POX application, and apoptotic rates of EL4 cells after treatment with POX for 16h were also increased in a dose-dependent manner. Pre-treatment with EGTA, heparin or procaine attenuated POX-induced [Ca(2+)]i elevation and apoptosis. Additionally, POX up-regulated caspase-12 expression in a dose-dependent manner, and pre-treatment with EGTA, heparin or procaine significantly inhibited POX-induced increase of caspase-12 expression. Our results suggested that POX induced [Ca(2+)]i elevation in EL4 cells at the early stage of POX-induced apoptosis, which might involve Ca(2+) efflux from the endoplasmic reticulum (ER) and Ca(2+) influx from extracellular medium. Calcium signals and caspase-12 were important upstream messengers in POX-induced apoptosis in EL4 cells. The ER-associated pathway possibly operated in this apoptosis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Influence of extra-cellular and intra-cellular acting thiol oxidants on the 45calcium uptake by the islets of Langerhans of the rat

International Nuclear Information System (INIS)

Haegele, R.G.

1981-01-01

The glucose-stimulated calcium uptake by the islets of Langerhans is dependent on the intra-cellular GSH/GSSG ratios. The inhibition of calcium uptake is not the consequence of a direct oxidation of membrane-fixed thiol groups. In contrast, direct oxidation of extra cellular thiols leads to an increase in calcium uptake when intra-cellular oxidation is simultaneously prevented. Since this effect only occurs at high intra-cellular GSH/GSSG ratios it can be assumed that the redox state of extra-cellular thiols is dependent on the redox state of the intra-cellular GSH/GSSG ratios. These findings support the theory that the oxidation of extra-cellular thiols by thiol oxidants leads to an increase in calcium uptake and that the extent of uptake is higher, the more the redox state of the extra-cellular thiols tends towards the reduced state prior to oxidation. (orig./MG) [de

Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis.

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López, Daniel; Kolter, Roberto

2010-03-01

The soil-dwelling bacterium Bacillus subtilis differentiates into distinct subpopulations of specialized cells that coexist within highly structured communities. The coordination and interplay between these cell types requires extensive extracellular communication driven mostly by sensing self-generated secreted signals. These extracellular signals activate a set of sensor kinases, which respond by phosphorylating three major regulatory proteins, Spo0A, DegU and ComA. Each phosphorylated regulator triggers a specific differentiation program while at the same time repressing other differentiation programs. This allows a cell to differentiate in response to a specific cue, even in the presence of other, possibly conflicting, signals. The sensor kinases involved respond to an eclectic group of extracellular signals, such as quorum-sensing molecules, natural products, temperature, pH or scarcity of nutrients. This article reviews the cascades of cell differentiation pathways that are triggered by sensing extracellular signals. We also present a tentative developmental model in which the diverse cell types sequentially differentiate to achieve the proper development of the bacterial community.

Autocrine signal transmission with extracellular ligand degradation

International Nuclear Information System (INIS)

Muratov, C B; Posta, F; Shvartsman, S Y

2009-01-01

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand–receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers

Autocrine signal transmission with extracellular ligand degradation

Science.gov (United States)

Muratov, C B; Posta, F; Shvartsman, S Y

2009-03-01

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand-receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers.

Extracellular Bio-imaging of Acetylcholine-stimulated PC12 Cells Using a Calcium and Potassium Multi-ion Image Sensor.

Science.gov (United States)

Matsuba, Sota; Kato, Ryo; Okumura, Koichi; Sawada, Kazuaki; Hattori, Toshiaki

2018-01-01

In biochemistry, Ca 2+ and K + play essential roles to control signal transduction. Much interest has been focused on ion-imaging, which facilitates understanding of their ion flux dynamics. In this paper, we report a calcium and potassium multi-ion image sensor and its application to living cells (PC12). The multi-ion sensor had two selective plasticized poly(vinyl chloride) membranes containing ionophores. Each region on the sensor responded to only the corresponding ion. The multi-ion sensor has many advantages including not only label-free and real-time measurement but also simultaneous detection of Ca 2+ and K + . Cultured PC12 cells treated with nerve growth factor were prepared, and a practical observation for the cells was conducted with the sensor. After the PC12 cells were stimulated by acetylcholine, only the extracellular Ca 2+ concentration increased while there was no increase in the extracellular K + concentration. Through the practical observation, we demonstrated that the sensor was helpful for analyzing the cell events with changing Ca 2+ and/or K + concentration.

Calcium signaling in smooth muscle.

Science.gov (United States)

Hill-Eubanks, David C; Werner, Matthias E; Heppner, Thomas J; Nelson, Mark T

2011-09-01

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

Calcium and ROS: A mutual interplay

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Görlach, Agnes; Bertram, Katharina; Hudecova, Sona; Krizanova, Olga

2015-01-01

Calcium is an important second messenger involved in intra- and extracellular signaling cascades and plays an essential role in cell life and death decisions. The Ca2+ signaling network works in many different ways to regulate cellular processes that function over a wide dynamic range due to the action of buffers, pumps and exchangers on the plasma membrane as well as in internal stores. Calcium signaling pathways interact with other cellular signaling systems such as reactive oxygen species (ROS). Although initially considered to be potentially detrimental byproducts of aerobic metabolism, it is now clear that ROS generated in sub-toxic levels by different intracellular systems act as signaling molecules involved in various cellular processes including growth and cell death. Increasing evidence suggests a mutual interplay between calcium and ROS signaling systems which seems to have important implications for fine tuning cellular signaling networks. However, dysfunction in either of the systems might affect the other system thus potentiating harmful effects which might contribute to the pathogenesis of various disorders. PMID:26296072

Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor.

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Kleinau, Gunnar; Jaeschke, Holger; Mueller, Sandra; Raaka, Bruce M; Neumann, Susanne; Paschke, Ralf; Krause, Gerd

2008-08-01

The mechanisms governing transition of the thyroid stimulating hormone (TSH) receptor (TSHR) from basal to active conformations are poorly understood. Considering that constitutively activating mutations (CAMs) and inactivating mutations in each of the extracellular loops (ECLs) trigger only partial TSHR activation or inactivation, respectively, we hypothesized that full signaling occurs via multiple extracellular signal propagation events. Therefore, individual CAMs in the extracellular region were combined to create double and triple mutants. In support of our hypothesis, combinations of mutants in the ECLs are in some cases additive, while in others they are even synergistic, with triple mutant I486A/I568V/V656F exhibiting a 70-fold increase in TSH-independent signaling. The proximity but likely different spatial orientation of the residues of activating and inactivating mutations in each ECL supports a dual functionality to facilitate signal induction and conduction, respectively. This is the first report for G-protein coupled receptors, suggesting that multiple and cooperative signal propagating events at all three ECLs are required for full receptor activation. Our findings provide new insights concerning molecular signal transmission from extracellular domains toward the transmembrane helix bundle of the glycoprotein hormone receptors.

Lipophilic Chemicals from Diesel Exhaust Particles Trigger Calcium Response in Human Endothelial Cells via Aryl Hydrocarbon Receptor Non-Genomic Signalling

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Bendik C. Brinchmann

2018-05-01

Full Text Available Exposure to diesel exhaust particles (DEPs affects endothelial function and may contribute to the development of atherosclerosis and vasomotor dysfunction. As intracellular calcium concentration [Ca2+]i is considered important in myoendothelial signalling, we explored the effects of extractable organic matter from DEPs (DEP-EOM on [Ca2+]i and membrane microstructure in endothelial cells. DEP-EOM of increasing polarity was obtained by pressurized sequential extraction of DEPs with n-hexane (n-Hex-EOM, dichloromethane (DCM-EOM, methanol, and water. Chemical analysis revealed that the majority of organic matter was extracted by the n-Hex- and DCM-EOM, with polycyclic aromatic hydrocarbons primarily occurring in n-Hex-EOM. The concentration of calcium was measured in human microvascular endothelial cells (HMEC-1 using micro-spectrofluorometry. The lipophilic n-Hex-EOM and DCM-EOM, but not the more polar methanol- and water-soluble extracts, induced rapid [Ca2+]i increases in HMEC-1. n-Hex-EOM triggered [Ca2+]i increase from intracellular stores, followed by extracellular calcium influx consistent with store operated calcium entry (SOCE. By contrast, the less lipophilic DCM-EOM triggered [Ca2+]i increase via extracellular influx alone, resembling receptor operated calcium entry (ROCE. Both extracts increased [Ca2+]i via aryl hydrocarbon receptor (AhR non-genomic signalling, verified by pharmacological inhibition and RNA-interference. Moreover, DCM-EOM appeared to induce an AhR-dependent reduction in the global plasma membrane order, as visualized by confocal fluorescence microscopy. DCM-EOM-triggered [Ca2+]i increase and membrane alterations were attenuated by the membrane stabilizing lipid cholesterol. In conclusion, lipophilic constituents of DEPs extracted by n-hexane and DCM seem to induce rapid AhR-dependent [Ca2+]i increase in HMEC-1 endothelial cells, possibly involving both ROCE and SOCE-mediated mechanisms. The semi-lipophilic fraction

A study on MR signal intensity of calcifications according to calcium concentration and compound

International Nuclear Information System (INIS)

Chang, Sun Ae; Kim, Jae Hyoung; Chung, Sung Hoon

1996-01-01

The signal of intracranial calcification on magnetic resonance (MR) imaging has been known to be variable. The purpose of this study was to evaluate the MR signal of calcifications according to calcium concentration and compound. T1-weighted, proton density and T2-weighted images were obtained in phantoms with various conposition of calcium carbonate and calcium phosphate. The signal intensities and T1/T2 relaxation times were measured and analyzed according to calcium concentration and compound. The configurations of calcium particles were evaluated by scanning electron microscopy. The signal intensity of calcium carbonate on T1-weighted images gradually decreased as the concentration increased, while that of calcium phosphate showed a biphasic curve with a peak intensity at 0.2g/ml. The signal intensity of both calcium phosphate decreased up to 0.2g/ml and then remained constant. The T2 relaxation time of both calcium compounds decreased in a similar fashion with increasing concentration, Calcium phosphate showed larger surface area on scanning electron microscope. Calcifications show variable MR signal due to difference of T1 and T2 relaxation times according to calcium concentration and compound. Large surface area of calcium particle might cause shortening of T1 relaxation time leading to high signal on T1-weighted image. Understanding of these findings will help interpretation of MR images more precisely

[Myofibroblasts and afferent signalling in the urinary bladder. A concept].

Science.gov (United States)

Neuhaus, J; Scholler, U; Freick, K; Schwalenberg, T; Heinrich, M; Horn, L C; Stolzenburg, J U

2008-09-01

Afferent signal transduction in the urinary bladder is still not clearly understood. An increasing body of evidence supports the view of complex interactions between urothelium, suburothelial myofibroblasts, and sensory nerves. Bladder tissue from tumour patients was used in this study. Methods included confocal immunofluorescence, polymerase chain reaction, calcium imaging, and fluorescence recovery after photobleaching (FRAP).Myofibroblasts express muscarinic and purinergic receptors. They show constitutive spontaneous activity in calcium imaging, which completely depends on extracellular calcium. Stimulation with carbachol and ATP-evoked intracellular calcium transients also depend on extracellular calcium. The intensive coupling between the cells is significantly diminished by incubation with TGF-beta 1. Myofibroblasts form an important cellular element within the afferent signalling of the urinary bladder. They possess all features required to take part in the complex interactions with urothelial cells and sensory nerves. Modulation of their function by cytokines may provide a pathomechanism for bladder dysfunction.

Lipopolysaccharide (LPS)-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity

International Nuclear Information System (INIS)

Drysdale, B.E.; Shin, H.S.

1986-01-01

As the authors reported, calcium ionophore, A23187, activates macrophages (M theta) for tumor cell killing and the activated M theta produce a soluble cytotoxic factor (M theta-CF) that is similar if not identical to tumor necrosis factor. Based on these observations they have investigated whether calcium is involved in the activation mediated by another potent M theta activator, LPS. The authors have shown that A23187 caused uptake of extracellular 45 Ca ++ but LPS did not. They have examined the effect of depleting extracellular calcium by using medium containing no added calcium containing 1.0 mM EGTA. In no case did depletion result in decreased M theta-CF production by the M theta activated with LPS. Measurements using the fluorescent, intracellular calcium indicator, Quin 2 have also been performed. While ionomycin, caused a rapid change in the Quin-2 signal, LPS at a concentration even in excess of that required to activate the M theta caused no change in the signal. When high doses of Quin 2 or another intracellular chelator, 8-(diethylaminol-octyl-3,4,5-trimethoxybenzoate, were used to treat M theta, M theta-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M theta-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium

The interplay between HIF-1 and calcium signalling in cancer.

Science.gov (United States)

Azimi, Iman

2018-04-01

The interplay between hypoxia-inducible factor-1 (HIF-1) and calcium in cancer has begun to be unravelled with recent findings demonstrating the relationships between the two in different cancer types. This is an area of significance considering the crucial roles of both HIF-1 and calcium signalling in cancer progression and metastasis. This review summarises the experimental evidence of the crosstalk between HIF-1 and specific calcium channels, pumps and regulators in the context of cancer. HIF-1 as a master regulator of hypoxic transcriptional responses, mediates transcription of several calcium modulators. On the other hand, specific calcium channels and pumps regulate HIF-1 activity through controlling its transcription, translation, stabilisation, or nuclear translocation. Identifying the interplay between HIF-1 and components of the calcium signal will give new insights into mechanisms underlying cellular responses to physiological and pathophysiological cues, and may provide novel and more efficient therapeutic strategies for the control of cancer progression. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

DEFF Research Database (Denmark)

Petersen, R H; Knudsen, T; Johansen, Torben

1991-01-01

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat ...

Cardiac voltage gated calcium channels and their regulation by β-adrenergic signaling.

Science.gov (United States)

Kumari, Neema; Gaur, Himanshu; Bhargava, Anamika

2018-02-01

Voltage-gated calcium channels (VGCCs) are the predominant source of calcium influx in the heart leading to calcium-induced calcium release and ultimately excitation-contraction coupling. In the heart, VGCCs are modulated by the β-adrenergic signaling. Signaling through β-adrenergic receptors (βARs) and modulation of VGCCs by β-adrenergic signaling in the heart are critical signaling and changes to these have been significantly implicated in heart failure. However, data related to calcium channel dysfunction in heart failure is divergent and contradictory ranging from reduced function to no change in the calcium current. Many recent studies have highlighted the importance of functional and spatial microdomains in the heart and that may be the key to answer several puzzling questions. In this review, we have briefly discussed the types of VGCCs found in heart tissues, their structure, and significance in the normal and pathological condition of the heart. More importantly, we have reviewed the modulation of VGCCs by βARs in normal and pathological conditions incorporating functional and structural aspects. There are different types of βARs, each having their own significance in the functioning of the heart. Finally, we emphasize the importance of location of proteins as it relates to their function and modulation by co-signaling molecules. Its implication on the studies of heart failure is speculated. Copyright © 2017 Elsevier Inc. All rights reserved.

Intracellular sphingosine releases calcium from lysosomes.

Science.gov (United States)

Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

2015-11-27

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

International Nuclear Information System (INIS)

Tong, P.; Mayes, D.; Wheeler, L.

1986-01-01

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca ++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca ++ medium (0.09 mM) as measured by incorporation of [ 3 H] thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC 50 of about 10 μM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 μM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number of control at 10 μM. These results are of importance since they suggest Ca ++ may influence the effect of retinoids on keratinocytes

Resveratrol and Calcium Signaling: Molecular Mechanisms and Clinical Relevance

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Audrey E. McCalley

2014-06-01

Full Text Available Resveratrol is a naturally occurring compound contributing to cellular defense mechanisms in plants. Its use as a nutritional component and/or supplement in a number of diseases, disorders, and syndromes such as chronic diseases of the central nervous system, cancer, inflammatory diseases, diabetes, and cardiovascular diseases has prompted great interest in the underlying molecular mechanisms of action. The present review focuses on resveratrol, specifically its isomer trans-resveratrol, and its effects on intracellular calcium signaling mechanisms. As resveratrol’s mechanisms of action are likely pleiotropic, its effects and interactions with key signaling proteins controlling cellular calcium homeostasis are reviewed and discussed. The clinical relevance of resveratrol’s actions on excitable cells, transformed or cancer cells, immune cells and retinal pigment epithelial cells are contrasted with a review of the molecular mechanisms affecting calcium signaling proteins on the plasma membrane, cytoplasm, endoplasmic reticulum, and mitochondria. The present review emphasizes the correlation between molecular mechanisms of action that have recently been identified for resveratrol and their clinical implications.

Short-range intercellular calcium signaling in bone

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye

2005-01-01

different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other...... to osteoclasts as well. We demonstrated that paracrine action of ATP was responsible for the wave propagation, but now the purinergic P2X7 receptor was involved. Thus, the studies demonstrate that calcium signals can be propagated not only among osteoblasts, but also between osteoblasts and osteoclasts...

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMs and CDPKs leading to copper entry and membrane depolarization in Ulva compressa

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Melissa eGómez

2015-03-01

Full Text Available In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1 and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9 and 12 min of exposure, respectively, and antagonists of TRPC5, A1 and V1 corresponding to SKF-96365 (SKF, HC-030031 (HC and capsazepin (CPZ, respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9 and 12 min which were inhibited by SKF, HC and CPZ, respectively, indicating that copper activate TRPC5, A1 and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require CaMs and CDPKs activation. In addition, copper induced membrane depolarization events at 4, 8 and 11 min and these events were inhibited by SKF, HC, CPZ and bathocuproine, a specific copper chelating agent, indicating copper entry through TRP channels leading to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that CaMs and CDPKs are required in order to activate TRPs to allow copper entry. Thus, light-dependent copper-induced activation TRPC5, A1 and V1 promotes extracellular calcium entry leading to activation of CaMs and CDPKs which, in turn, promotes copper entry through these TRP channels leading to membrane depolarization.

Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium.

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Aggarwal, Abhishek; Schulz, Herbert; Manhardt, Teresa; Bilban, Martin; Thakker, Rajesh V; Kallay, Enikö

2017-06-01

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca 2+ ] o ) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca 2+ ] o identified significant changes in expression of 1571 probe sets (ANOVA, pcalcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca 2+ ] o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Lrp4: a novel modulator of extracellular signaling in craniofacial organogenesis

OpenAIRE

Ohazama, Atsushi; Porntaveetus, Thantrira; Ota, Masato S.; Herz, Joachim; Sharpe, Paul T.

2010-01-01

The low-density lipoprotein (LDL) receptor family is a large evolutionarily conserved group of transmembrane proteins. It has been shown that LDL receptor family members can also function as direct signal transducers or modulators for a broad range of cellular signalling pathways. We have identified a novel mode of signalling pathway integration/coordination that occurs outside cells during development that involves an LDL family member. Physical interaction between an extracellular protein (...

Induced effect of Ca2+ on dalesconols A and B biosynthesis in the culture of Daldinia eschscholzii via calcium/calmodulin signaling.

Science.gov (United States)

Lu, Yanhua; Pan, Zhenghua; Tao, Jun; An, Faliang

2018-02-01

Dalesconols (dalesconols A and B) were isolated from Daldinia eschscholzii and have remarkable immunosuppressive activity. In this study, the response of fungal growth, intra- and extracellular Ca 2+ , and dalesconols production after CaCl 2 addition were reported for the first time. After supplementation with 5 mM Ca 2+ at 24 h, dalesconols production reached 84.33 mg/L, which resulted in a 1.57-fold enhancement compared to the control. The key role of calcium/calmodulin signaling in dalesconols biosynthesis was confirmed by treatment with Ca 2+ channel and calmodulin inhibitors. The transcriptional levels of dalesconols biosynthetic genes were up-regulated after CaCl 2 addition and down-regulated after inhibitors were added. The results demonstrated that Ca 2+ addition induces dalesconols biosynthesis through up-regulation of dalesconols biosynthesis genes via regulation of calcium/calmodulin signaling. This study provided an efficient strategy for improving dalesconols production and would facilitate further research on the biosynthesis and regulation of dalesconols. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

DEFF Research Database (Denmark)

Kowal, Justyna Magdalena

release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological......ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......, particularly during Ca2+ stress conditions. In conclusion, these studies demonstrate a complex regulation of purinergic signalling in exocrine pancreas. A crucial role for duct cells in mediating extracellular nucleotides homeostasis, involving ATP release, subsequent hydrolysis and conversion via...

Extracellular ATP acts as a damage associated molecular pattern (DAMP signal in plants

Directory of Open Access Journals (Sweden)

Kiwamu eTanaka

2014-09-01

Full Text Available As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs. ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling role in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor Kinase, which is plant-specific. P2K1 (DORN1 is required for ATP-induced cellular responses (e.g., cytosolic Ca2+ elevation, MAPK phosphorylation, and gene expression. Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of the future research of extracellular ATP as a DAMP signal.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

Science.gov (United States)

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

Science.gov (United States)

Kishore, Ayush; Hall, Randy A

2017-06-09

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient Extracellular Expression of Phospholipase D in Escherichia Coli with an Optimized Signal Peptide

Science.gov (United States)

Yang, Leyun; Xu, Yu; Chen, Yong; Ying, Hanjie

2018-01-01

New secretion vectors containing the synthetic signal sequence (OmpA’) was constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli Phospholipase D structural gene (Accession number:NC_018658) fused to various signal sequence were expressed from the Lac promoter in E. coli Rosetta strains by induction with 0.4mM IPTG at 28°C for 48h. SDS-PaGe analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of Phospholipase D (PLD) not only to the medium but also remained in periplasm of E. coli with OmpA’ signal sequence at the N-terminus of PLD. Thus the study on the effects of various surfactants on PLD extracellular production in Escherichia coli in shake flasks revealed that optimal PLD extracellular production could be achieved by adding 0.4% Triton X-100 into the medium. The maximal extracellular PLD production and extracellular enzyme activity were 0.23mg ml-1 and 16U ml-1, respectively. These results demonstrate the possibility of efficient secretory production of recombinant PLD in E. coli should be a potential industrial applications.

Paclitaxel Induces Apoptosis in Breast Cancer Cells through Different Calcium—Regulating Mechanisms Depending on External Calcium Conditions

Science.gov (United States)

Pan, Zhi; Avila, Andrew; Gollahon, Lauren

2014-01-01

Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis. PMID:24549172

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

Science.gov (United States)

Vőfély, Gergő; Berecz, Tünde; Szabó, Eszter; Szebényi, Kornélia; Hathy, Edit; Orbán, Tamás I; Sarkadi, Balázs; Homolya, László; Marchetto, Maria C; Réthelyi, János M; Apáti, Ágota

2018-04-01

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Nuclear calcium signaling induces expression of the synaptic organizers Lrrtm1 and Lrrtm2.

Science.gov (United States)

Hayer, Stefanie N; Bading, Hilmar

2015-02-27

Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2-4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca(2+)/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Nuclear Calcium Signaling Induces Expression of the Synaptic Organizers Lrrtm1 and Lrrtm2*

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Hayer, Stefanie N.; Bading, Hilmar

2015-01-01

Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2–4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca2+/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2. PMID:25527504

The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

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Assas, Bakri M; Abdulaal, Wesam H; Wakid, Majed H; Zakai, Haytham A; Miyan, J; Pennock, J L

2017-06-15

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment. Copyright © 2017. Published by Elsevier Inc.

Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

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Shankaran, Harish; Wiley, H. S.

2010-12-01

The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

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Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Lucchi, Chiara; Correia-de-Sá, Paulo; Videira, Arnaldo

2015-10-01

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics. Copyright © 2015 Elsevier B.V. All rights reserved.

Angiotensin II induces calcium/calcineurin signaling and podocyte injury by downregulating microRNA-30 family members.

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Zhao, Yue; Wu, Junnan; Zhang, Mingchao; Zhou, Minlin; Xu, Feng; Zhu, Xiaodong; Zhou, Xianguang; Lang, Yue; Yang, Fan; Yun, Shifeng; Shi, Shaolin; Liu, Zhihong

2017-08-01

Angiotensin II (AngII) is capable of inducing calcium/calcineurin signaling and podocyte injury; however, the precise underlying mechanism is not well understood. Because we have previously demonstrated that microRNA-30s (miR-30s) inhibit calcium/calcineurin signaling in podocytes, we hypothesize that AngII may induce podocyte injury by downregulating miR-30s and thereby activating calcium/calcineurin signaling. To test this hypothesis, we used an AngII-induced podocyte injury mouse model. The mice were treated with AngII via infusion for 28 days, which resulted in hypertension, albuminuria, and glomerular damage. AngII treatment also resulted in a significant reduction of miR-30s and upregulation of calcium/calcineurin signaling components, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, which are the known targets of miR-30s in podocytes. The delivery of miR-30a-expressing lentivirus to the podocytes on day 14 of the infusion ameliorated the AngII-induced podocyte and glomerular injury and attenuated the upregulation of the calcium/calcineurin signaling components. Similarly, treatment with losartan, which is an AngII receptor blocker, also prevented AngII-induced podocyte injury and calcium/calcineurin signaling activation. Notably, losartan was found to sustain miR-30 levels during AngII treatment both in vivo and in vitro. In conclusion, the effect of AngII on podocytes is in part mediated by miR-30s through calcium/calcineurin signaling, a novel mechanism underlying AngII-induced podocyte injury. • AngII infusion resulted in downregulation of miR-30s in podocytes. • Exogenous miR-30a delivery mitigated the glomerular and podocyte injuries induced by AngII. • Both miR-30a and losartan prevented AngII-induced activation of calcium-calcineurin signaling.

The role of calcium-sensing receptor and signalling pathways in the pathophysiology in two in vitro models of malignant hypercalcemia: the rat rice H-500 Leydig testis cancer and prostate cancer (PC-3) cells. Expression and regulation of pituitary tumor transforming gene in Leydig testis cancer

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Tfelt-Hansen, J.

2008-01-01

The calcium-sensing receptor (CaR) is a seven transmembrane receptor incorporated into the cell membrane that is sensitive to extracellular calcium and other cations. The finding that the CaR is expressed on cancer cells has opened the door to a new understanding of the role of extracellular...... too many adverse effects on calcium homeostasis. In conclusion, the CaR plays diverse roles in cancer-acting as an inhibitor of cell proliferation in the colon crypt cells giving rise to colon cancer but as a promalignant receptor in most other cancer types, including Leydig cell cancers...... calcium as a promalignant stimulus through the CaR and its signaling apparatus as demonstrated in this thesis. I found, in a model of humoral hypercalcemia of malignancy (HHM), that stimulation of the CaR worsens the promalignant features of the testicular H-500 Leydig cancer cells that were used in my...

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

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Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Amino alcohol- (NPS-2143 and quinazolinone-derived calcilytics (ATF936 and AXT914 differentially mitigate excessive signalling of calcium-sensing receptor mutants causing Bartter syndrome Type 5 and autosomal dominant hypocalcemia.

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Saskia Letz

Full Text Available Activating calcium sensing receptor (CaSR mutations cause autosomal dominant hypocalcemia (ADH characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics on activating CaSR mutants.All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o. To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914.All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.

A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

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Christine Dorothee Schmeitz

2013-09-01

Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

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Schultz, Robbie D; Bennett, Emily E; Ellis, E Ann; Gumienny, Tina L

2014-01-01

In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

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Robbie D Schultz

Full Text Available In mammals, Bone Morphogenetic Protein (BMP pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

Intercellular calcium signaling is regulated by morphogens during Drosophila wing development

OpenAIRE

Chen, Danny; Levis, Megan; Arredondo-Walsh, Ninfamaria; Zartman, Jeremiah; Brodskiy, Pavel; Wu, Qinfeng; Huizar, Francisco; Soundarrajan, Dharsan; Narciso, Cody; Chen, Jianxu; Liang, Peixian

2017-01-01

Organ development is driven by a set of patterned inductive signals. However, how these signals are integrated to coordinate tissue patterning is still poorly understood. Calcium ions (Ca2+) are critical signaling components involved in signal integration and are regulated by a core Ca2+ signaling toolkit. Ca2+ signaling encodes a significant fraction of information in cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. A range ...

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

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Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

2013-09-01

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

Silver Nanoparticle-Directed Mast Cell Degranulation Is Mediated through Calcium and PI3K Signaling Independent of the High Affinity IgE Receptor.

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Nasser B Alsaleh

Full Text Available Engineered nanomaterial (ENM-mediated toxicity often involves triggering immune responses. Mast cells can regulate both innate and adaptive immune responses and are key effectors in allergic diseases and inflammation. Silver nanoparticles (AgNPs are one of the most prevalent nanomaterials used in consumer products due to their antimicrobial properties. We have previously shown that AgNPs induce mast cell degranulation that was dependent on nanoparticle physicochemical properties. Furthermore, we identified a role for scavenger receptor B1 (SR-B1 in AgNP-mediated mast cell degranulation. However, it is completely unknown how SR-B1 mediates mast cell degranulation and the intracellular signaling pathways involved. In the current study, we hypothesized that SR-B1 interaction with AgNPs directs mast cell degranulation through activation of signal transduction pathways that culminate in an increase in intracellular calcium signal leading to mast cell degranulation. For these studies, we utilized bone marrow-derived mast cells (BMMC isolated from C57Bl/6 mice and RBL-2H3 cells (rat basophilic leukemia cell line. Our data support our hypothesis and show that AgNP-directed mast cell degranulation involves activation of PI3K, PLCγ and an increase in intracellular calcium levels. Moreover, we found that influx of extracellular calcium is required for the cells to degranulate in response to AgNP exposure and is mediated at least partially via the CRAC channels. Taken together, our results provide new insights into AgNP-induced mast cell activation that are key for designing novel ENMs that are devoid of immune system activation.

Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

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Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

2013-11-15

Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses. © 2013 Published by Elsevier B.V.

Composite mathematical modeling of calcium signaling behind neuronal cell death in Alzheimer's disease.

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Ranjan, Bobby; Chong, Ket Hing; Zheng, Jie

2018-04-11

Alzheimer's disease (AD) is a progressive neurological disorder, recognized as the most common cause of dementia affecting people aged 65 and above. AD is characterized by an increase in amyloid metabolism, and by the misfolding and deposition of β-amyloid oligomers in and around neurons in the brain. These processes remodel the calcium signaling mechanism in neurons, leading to cell death via apoptosis. Despite accumulating knowledge about the biological processes underlying AD, mathematical models to date are restricted to depicting only a small portion of the pathology. Here, we integrated multiple mathematical models to analyze and understand the relationship among amyloid depositions, calcium signaling and mitochondrial permeability transition pore (PTP) related cell apoptosis in AD. The model was used to simulate calcium dynamics in the absence and presence of AD. In the absence of AD, i.e. without β-amyloid deposition, mitochondrial and cytosolic calcium level remains in the low resting concentration. However, our in silico simulation of the presence of AD with the β-amyloid deposition, shows an increase in the entry of calcium ions into the cell and dysregulation of Ca 2+ channel receptors on the Endoplasmic Reticulum. This composite model enabled us to make simulation that is not possible to measure experimentally. Our mathematical model depicting the mechanisms affecting calcium signaling in neurons can help understand AD at the systems level and has potential for diagnostic and therapeutic applications.

Calcium carbonate formation on mica supported extracellular polymeric substance produced by Rhodococcus opacus

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Szcześ, Aleksandra, E-mail: aszczes@poczta.umcs.lublin.pl [Department of Physical Chemistry – Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Skłodowska University, Lublin 20-031 (Poland); Czemierska, Magdalena; Jarosz-Wilkołazka, Anna [Department of Biochemistry, Maria Curie-Skłodowska University, Lublin 20-031 (Poland)

2016-10-15

Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitated CaCO{sub 3} polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions. - Highlights: • CaCO{sub 3} crystal size and polymorph can be controlled by EPS substance obtained from R. opacus. • The water soluble fraction favours vaterite dissolution and calcite growth. • The total EPS stabilizes vaterite. • This effect is stronger at basic pH.

Calcium carbonate formation on mica supported extracellular polymeric substance produced by Rhodococcus opacus

International Nuclear Information System (INIS)

Szcześ, Aleksandra; Czemierska, Magdalena; Jarosz-Wilkołazka, Anna

2016-01-01

Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitated CaCO 3 polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions. - Highlights: • CaCO 3 crystal size and polymorph can be controlled by EPS substance obtained from R. opacus. • The water soluble fraction favours vaterite dissolution and calcite growth. • The total EPS stabilizes vaterite. • This effect is stronger at basic pH.

Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

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Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

2011-01-01

Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

The Acid Test: Calcium Signaling in the Skeletogenic Layer of Reef-Building Coral

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Florn, A. M.

2016-02-01

Since the Industrial Revolution, carbon dioxide (CO2) emissions have increased more than 40%. This increased atmospheric CO2 drives ocean acidification and has potentially serious consequences for all marine life, especially calcifying organisms. The specific goal of this study was to examine calcium homeostasis and signaling dynamics within the skeletogenic tissue layers (calicodermal cells) of two coral species (Pavona maldivensis and Porites rus) at three pH treatments corresponding to present-future ocean acidification levels. Confocal microscopy techniques were used to analyze in vivo calcium dynamics of the calicodermal cells in Pavona maldivensis and Porites rus. The results show biological variation between the two reef-building coral species and their response to ocean acidification. Pavona maldivensis showed a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments, but not among the microcolonies. Porites rus did not show a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments or the microcolonies. Upon comparing the calcium response curves, the ionomycin-induced calcium response exhibited by Pavona maldivensis is phenomenologically similar to a calcium response that is commonly found in vertebrates. This well-studied phenomenon in vertebrate biology is known as store-operated calcium entry (SOCE) and is closely associated with the endoplasmic reticulum (ER) and mitochondria-associated endoplasmic reticulum (MAM) calcium stores. This study provides insight into the preliminary steps needed to understand in vivo calcium signaling in the calicodermis of reef-building coral and the associated consequences of ocean acidification.

Calcium efflux systems in stress signalling and adaptation in plants

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Jayakumar eBose

2011-12-01

Full Text Available Transient cytosolic calcium ([Ca2+]cyt elevation is an ubiquitous denominator of the signalling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium signature that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms in shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signalling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analysed in detail. The spatial and temporal organisation of calcium signalling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarised. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modelled by using a four-component model (plasma- and endo- membrane-based Ca2+-permeable channels and efflux systems taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasising the crucial role these active efflux systems play in plant adaptive responses to environment.

Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc

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MP Grant

2016-07-01

Full Text Available The cartilaginous endplates (CEPs are thin layers of hyaline cartilage found adjacent to intervertebral discs (IVDs. In addition to providing structural support, CEPs regulate nutrient and metabolic exchange in the disc. In IVD pathogenesis, CEP undergoes degeneration and calcification, compromising nutrient availability and disc cell metabolism. The mechanism(s underlying the biochemical changes of CEP in disc degeneration are currently unknown. Since calcification is often observed in later stages of IVD degeneration, we hypothesised that elevations in free calcium (Ca2+ impair CEP homeostasis. Indeed, our results demonstrated that the Ca2+ content was consistently higher in human CEP tissue with grade of disc degeneration. Increasing the levels of Ca2+ resulted in decreases in the secretion and accumulation of collagens type I, II and proteoglycan in cultured human CEP cells. Ca2+ exerted its effects on CEP matrix protein synthesis through activation of the extracellular calcium-sensing receptor (CaSR; however, aggrecan content was also affected independent of CaSR activation as increases in Ca2+ directly enhanced the activity of aggrecanases. Finally, supplementing Ca2+ in our IVD organ cultures was sufficient to induce degeneration and increase the mineralisation of CEP, and decrease the diffusion of glucose into the disc. Thus, any attempt to induce anabolic repair of the disc without addressing Ca2+ may be impaired, as the increased metabolic demand of IVD cells would be compromised by decreases in the permeability of the CEP.

Dual functional extracellular recording using a light-addressable potentiometric sensor for bitter signal transduction.

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Du, Liping; Wang, Jian; Chen, Wei; Zhao, Luhang; Wu, Chunsheng; Wang, Ping

2018-08-31

This paper presents a dual functional extracellular recording biosensor based on a light-addressable potentiometric sensor (LAPS). The design and fabrication of this biosensor make it possible to record both extracellular membrane potential changes and ATP release from a single taste bud cell for the first time. For detecting ATP release, LAPS chip was functionalized with ATP-sensitive DNA aptamer by covalent immobilization. Taste bud cells isolated from rat were cultured on LAPS surface. When the desired single taste bud cell was illuminated by modulated light, ATP release from single taste bud cells can be measured by recording the shifts of bias voltage-photocurrent curves (I-V curves) when the LAPS chip is working in discrete mode. On the other hand, extracellular membrane potential changes can be monitored by recording the fluctuation of LAPS photocurrent when the LAPS chip is working in continuous mode. The results show this biosensor can effectively record the enhancive effect of the bitter substance and inhibitory effect of the carbenoxolone (CBX) on the extracellular membrane potential changes and ATP release of single taste bud cells. In addition, the inhibitory effect of CBX also confirms LAPS extracellular recordings are originated from bitter signal transduction. It is proved this biosensor is suitable for extracellular recording of ATP release and membrane potential changes of single taste bud cells. It is suggested this biosensor could be applied to investigating taste signal transduction at the single-cell level as well as applied to other types of cells which have similar functions to taste bud cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Potassium conductances mediate bidirectional state-dependent modulation of action potential evoked dendritic calcium signals in dentate gyrus granule cells

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János Brunner

2014-03-01

Full Text Available Backpropagating action potentials (bAPs and local calcium signals that they trigger are fundamental for dendritic functions. Here we addressed the question what extent the changes of local dendritic membrane properties can contribute to the shaping of the coupling between dendritic action potentials and the local calcium responses. Using a combination of in vitro electrophysiological and confocal imaging techniques we found that activation of dendritic GIRK channels via mGlu2 or GABAB receptors enhanced the bAP¬-triggered calcium signals in the dendrites of dentate gyrus granule cells (GCs. The enhancement of calcium signals was significant only in those dendritic regions, where these receptors are predominantly expressed. Similarly to GIRK channel activation, somatic hyperpolarization by DC current injection (from -64 mV to -77 mV, significantly increased bAP-associated calcium signals in the proximal dendrites. The hyperpolarization was associated with a decrease in the input resistance due to the rectification of the membrane potential of GCs. The effect of hyperpolarization on the calcium signals was maintained when T-type calcium currents were blocked but it decreased when GIRK channels were inhibited. Simultaneous dual somato-dendritic recordings from GCs showed that somatic hyperpolarization accelerated the repolarization phase of dendritic bAP in the proximal region whereas the rising phase and peak amplitude was not affected. We hypothesize that the larger driving force for calcium ions during the faster repolarization can contribute to the increasing in calcium signals. Employment of previously recorded dendritic bAP waveforms from hyperpolarized membrane potential as voltage command evoked larger calcium currents in nucleated patches compared to bAP waveform from the same recording at depolarized membrane potential. Furthermore, addition of native, high-voltage activated, inactivating potassium conductance by somatic dynamic clamp

Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

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Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

2016-01-01

The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton.

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Pritchard, Scott; Erickson, Geoffrey R; Guilak, Farshid

2002-11-01

Loading of the spine alters the osmotic environment in the intervertebral disk (IVD) as interstitial water is expressed from the tissue. Cells from the three zones of the IVD, the anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP), respond to osmotic stress with altered biosynthesis through a pathway that may involve calcium (Ca(2+)) as a second messenger. We examined the hypothesis that IVD cells respond to hyperosmotic stress by increasing the concentration of intracellular calcium ([Ca(2+)](i)) through a mechanism involving F-actin. In response to hyperosmotic stress, control cells from all zones decreased in volume and cells from the AF and TZ exhibited [Ca(2+)](i) transients, while cells from the NP did not. Extracellular Ca(2+) was necessary to initiate [Ca(2+)](i) transients. Stabilization of F-actin with phalloidin prevented the Ca(2+) response in AF and TZ cells and decreased the rate of volume change in cells from all zones, coupled with an increase in the elastic moduli and apparent viscosity. Conversely, actin breakdown with cytochalasin D facilitated Ca(2+) signaling while decreasing the elastic moduli and apparent viscosity for NP cells. These results suggest that hyperosmotic stress induces volume change in IVD cells and may initiate [Ca(2+)](i) transients through an actin-dependent mechanism.

Filamin and phospholipase C-ε are required for calcium signaling in the Caenorhabditis elegans spermatheca.

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Ismar Kovacevic

2013-05-01

Full Text Available The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

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Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Bcl-2 overexpression: effects on transmembrane calcium movement

International Nuclear Information System (INIS)

Rangaswami, Arun A.; Premack, Brett; Walleczek, Jan; Killoran, Pamela; Gardner, Phyllis; Knox, Susan J.

1996-01-01

Purpose/Objective: High levels of expression of the proto-oncogene bcl-2 and its 26 kD protein product Bcl-2 have been correlated with the inhibition of apoptosis and the increased resistance of tumor cells to cytotoxic drugs and ionizing radiation. Unfortunately, the specific mechanism of action of Bcl-2 remains poorly understood. In the studies described here, the role of intracellular calcium fluxes and plasma membrane calcium cycling in the induction of apoptosis, and the effect of Bcl-2 expression on the modulation of transmembrane calcium fluxes following treatment of cells with cytotoxic agents were studied. The relationship between intracellular calcium release, capacitive calcium entry, and the plasma membrane potential were also investigated. Materials and Methods: Human B-cell lymphoma (PW) and human promyelocytic leukemia (HL60) cell lines were transfected with Bcl-2 and a control vector. The Bcl-2 transfectants over expressed the Bcl-2 onco-protein and were more resistant to irradiation than the control cells. Cells were loaded with fluorescent indicators indo-1 and fura-2 AM to quantify the cytosolic calcium concentration and subsequent calcium responses to a variety of cytotoxic stimuli, including the microsomal ATPase inhibitor, thapsigargin, using fluorometric measurements. Comparisons of resting and stimulated cytosolic calcium concentrations were made between the parental, neomycin control, and bcl-2 transfected cells. In order to determine the actual calcium influx rate, cells were loaded with either indo-1 or fura-2 and then exposed to 0.1 mM extracellular manganese, which enters the cells through calcium influx channels and quenches the fluorescent signal in proportion to the calcium influx rate. In order to determine the role of the membrane potential in driving calcium influx, cells were treated with either 0.1 μM Valinomycin or isotonic potassium chloride to either hyper polarize or depolarize the resting membrane potential, and the

AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

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Xia Luo

2015-01-01

Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

Visualization of Plasticity in Fear-Evoked Calcium Signals in Midbrain Dopamine Neurons

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Gore, Bryan B.; Soden, Marta E.; Zweifel, Larry S.

2014-01-01

Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium…

Odorant receptors directly activate phospholipase C/inositol-1,4,5-trisphosphate coupled to calcium influx in Odora cells.

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Liu, Guang; Badeau, Robert M; Tanimura, Akihiko; Talamo, Barbara R

2006-03-01

Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5-trisphosphate (IP(3)), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP(3) receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system.

Extracellular Calcium and Magnesium in Preeclampsia and Eclampsia

African Journals Online (AJOL)

The cause of preeclampsia remains unknown and calcium and magnesium supplement are being suggested as means of prevention. The objective of this study was to assess magnesium and calcium in the plasma and cerebrospinal fluid of Nigerian women with preeclampsia and eclampsia. Setting was University of ...

Calcium-sensing receptor (CaSR): pharmacological properties and signaling pathways.

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Conigrave, Arthur D; Ward, Donald T

2013-06-01

In this article we consider the mechanisms by which the calcium-sensing receptor (CaSR) induces its cellular responses via the control (activation or inhibition) of signaling pathways. We consider key features of CaSR-mediated signaling including its control of the heterotrimeric G-proteins Gq/11, Gi/o and G12/13 and the downstream consequences recognizing that very few CaSR-mediated cell phenomena have been fully described. We also consider the manner in which the CaSR contributes to the formation of specific signaling scaffolds via peptide recognition sequences in its intracellular C-terminal along with the origins of its high level of cooperativity, particularly for Ca(2+)o, and its remarkable resistance to desensitization. We also consider the nature of the mechanisms by which the CaSR controls oscillatory and sustained Ca(2+)i mobilizing responses and inhibits or elevates cyclic adenosine monophosphate (cAMP) levels dependent on the cellular and signaling context. Finally, we consider the diversity of the receptor's ligands, ligand binding sites and broader compartment-dependent physiological roles leading to the identification of pronounced ligand-biased signaling for agonists including Sr(2+) and modulators including l-amino acids and the clinically effective calcimimetic cinacalcet. We note the implications of these findings for the development of new designer drugs that might target the CaSR in pathophysiological contexts beyond those established for the treatment of disorders of calcium metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

Redox Signaling in Diabetic Wound Healing Regulates Extracellular Matrix Deposition.

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Kunkemoeller, Britta; Kyriakides, Themis R

2017-10-20

Impaired wound healing is a major complication of diabetes, and can lead to development of chronic foot ulcers in a significant number of patients. Despite the danger posed by poor healing, very few specific therapies exist, leaving patients at risk of hospitalization, amputation, and further decline in overall health. Recent Advances: Redox signaling is a key regulator of wound healing, especially through its influence on the extracellular matrix (ECM). Normal redox signaling is disrupted in diabetes leading to several pathological mechanisms that alter the balance between reactive oxygen species (ROS) generation and scavenging. Importantly, pathological oxidative stress can alter ECM structure and function. There is limited understanding of the specific role of altered redox signaling in the diabetic wound, although there is evidence that ROS are involved in the underlying pathology. Preclinical studies of antioxidant-based therapies for diabetic wound healing have yielded promising results. Redox-based therapeutics constitute a novel approach for the treatment of wounds in diabetes patients that deserve further investigation. Antioxid. Redox Signal. 27, 823-838.

Calcium homeostasis and signaling in fungi and their relevance for pathogenicity of yeasts and filamentous fungi

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Renata Tisi

2016-09-01

Full Text Available Though fungi show peculiarities in the purposes and specific traits of calcium signaling pathways, the general scheme and the most important players are well conserved if compared to higher eukaryotes. This provides a powerful opportunity either to investigate shared features using yeast as a model or to exploit fungal specificities as potential targets for antifungal therapies. The sequenced genomes from yeast Saccharomyces cerevisiae, Schizosaccharomyces pombe and the filamentous fungus Neurospora crassa were already published more than ten years ago. More recently the genome sequences of filamentous fungi of Aspergillus genus, some of which threatening pathogens, and dimorphic fungi Ustilago maydis were published, giving the chance to identify several proteins involved in calcium signaling based on their homology to yeast or mammalian counterparts. Nonetheless, unidentified calcium transporters are still present in these organisms which await to be molecularly characterized. Despite the relative simplicity in yeast calcium machinery and the availability of sophisticated molecular tools, in the last years, a number of new actors have been identified, albeit not yet fully characterized. This review will try to describe the state of the art in calcium channels and calcium signaling knowledge in yeast, with particular attention to the relevance of this knowledge with respect to pathological fungi.

Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

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Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; Nguyen, Desiree; Yong, Taiming; Yang, Paul G; Poretsky, Elly; Belknap, Thomas F; Waadt, Rainer; Alemán, Fernando; Schroeder, Julian I

2015-01-01

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level. DOI: http://dx.doi.org/10.7554/eLife.03599.001 PMID:26192964

Crosslink between calcium and sodium signalling.

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Verkhratsky, Alexei; Trebak, Mohamed; Perocchi, Fabiana; Khananshvili, Daniel; Sekler, Israel

2018-02-01

What is the topic of this review? This paper overviews the links between Ca 2+ and Na + signalling in various types of cells. What advances does it highlight? This paper highlights the general importance of ionic signalling and overviews the molecular mechanisms linking Na + and Ca 2+ dynamics. In particular, the narrative focuses on the molecular physiology of plasmalemmal and mitochondrial Na + -Ca 2+ exchangers and plasmalemmal transient receptor potential channels. Functional consequences of Ca 2+ and Na + signalling for co-ordination of neuronal activity with astroglial homeostatic pathways fundamental for synaptic transmission are discussed. Transmembrane ionic gradients, which are an indispensable feature of life, are used for generation of cytosolic ionic signals that regulate a host of cellular functions. Intracellular signalling mediated by Ca 2+ and Na + is tightly linked through several molecular pathways that generate Ca 2+ and Na + fluxes and are in turn regulated by both ions. Transient receptor potential (TRP) channels bridge endoplasmic reticulum Ca 2+ release with generation of Na + and Ca 2+ currents. The plasmalemmal Na + -Ca 2+ exchanger (NCX) flickers between forward and reverse mode to co-ordinate the influx and efflux of both ions with membrane polarization and cytosolic ion concentrations. The mitochondrial calcium uniporter channel (MCU) and mitochondrial Na + -Ca 2+ exchanger (NCLX) mediate Ca 2+ entry into and release from this organelle and couple cytosolic Ca 2+ and Na + fluctuations with cellular energetics. Cellular Ca 2+ and Na + signalling controls numerous functional responses and, in the CNS, provides for fast regulation of astroglial homeostatic cascades that are crucial for maintenance of synaptic transmission. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

The impact of mitochondrial endosymbiosis on the evolution of calcium signaling.

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Blackstone, Neil W

2015-03-01

At high concentrations, calcium has detrimental effects on biological systems. Life likely arose in a low calcium environment, and the first cells evolved mechanisms to maintain this environment internally. Bursts of calcium influx followed by efflux or sequestration thus developed in a functional context. For example, in proto-cells with exterior energy-converting membranes, such bursts could be used to depolarize the membrane. In this way, proto-cells could maintain maximal phosphorylation (metabolic state 3) and moderate levels of reactive oxygen species (ROS), while avoiding the resting state (metabolic state 4) and high levels of ROS. This trait is likely a shared primitive characteristic of prokaryotes. When eukaryotes evolved, the α-proteobacteria that gave rise to proto-mitochondria inhabited a novel environment, the interior of the proto-eukaryote that had a low calcium concentration. In this environment, metabolic homeostasis was difficult to maintain, and there were inherent risks from ROS, yet depolarizing the proto-mitochondrial membrane by calcium influx was challenging. To maintain metabolic state 3, proto-mitochondria were required to congregate near calcium influx points in the proto-eukaryotic membrane. This behavior, resulting in embryonic forms of calcium signaling, may have occurred immediately after the initiation of the endosymbiosis. Along with ROS, calcium may have served as one of the key forms of crosstalk among the community of prokaryotes that led to the eukaryotic cell. Copyright © 2014 Elsevier Ltd. All rights reserved.

Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins

Energy Technology Data Exchange (ETDEWEB)

Sirvent, P., E-mail: pascal.sirvent@univ-bpclermont.fr [U1046, INSERM, Université Montpellier 1 and Université Montpellier 2, 34295 Montpellier (France); CHRU Montpellier, 34295 Montpellier (France); Clermont Université, Université Blaise Pascal, EA 3533, Laboratoire des Adaptations Métaboliques à l' Exercice en conditions Physiologiques et Pathologiques (AME2P), BP 80026, F-63171 Aubière cedex (France); Fabre, O.; Bordenave, S. [U1046, INSERM, Université Montpellier 1 and Université Montpellier 2, 34295 Montpellier (France); CHRU Montpellier, 34295 Montpellier (France); Hillaire-Buys, D. [CHRU Montpellier, 34295 Montpellier (France); Raynaud De Mauverger, E.; Lacampagne, A.; Mercier, J. [U1046, INSERM, Université Montpellier 1 and Université Montpellier 2, 34295 Montpellier (France); CHRU Montpellier, 34295 Montpellier (France)

2012-03-01

The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro. -- Highlights: ► The most common and problematic side effect of statins is myopathy. ► Patients treated with statins showed impairment of mitochondrial respiration. ► Statins-treated patients showed altered frequency and amplitude of calcium sparks.

Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins

International Nuclear Information System (INIS)

Sirvent, P.; Fabre, O.; Bordenave, S.; Hillaire-Buys, D.; Raynaud De Mauverger, E.; Lacampagne, A.; Mercier, J.

2012-01-01

The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro. -- Highlights: ► The most common and problematic side effect of statins is myopathy. ► Patients treated with statins showed impairment of mitochondrial respiration. ► Statins-treated patients showed altered frequency and amplitude of calcium sparks.

Quantitative properties and receptor reserve of the IP(3) and calcium branch of G(q)-coupled receptor signaling.

Science.gov (United States)

Dickson, Eamonn J; Falkenburger, Björn H; Hille, Bertil

2013-05-01

Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5'-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition.

Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

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Alonso, Carlos Agustín I; Osycka-Salut, Claudia E; Castellano, Luciana; Cesari, Andreína; Di Siervi, Nicolás; Mutto, Adrián; Johannisson, Anders; Morrell, Jane M; Davio, Carlos; Perez-Martinez, Silvina

2017-08-01

Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA

Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry.

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Doll, Caleb A; Broadie, Kendal

2016-05-01

Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early

Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation.

Science.gov (United States)

Deymier, P A; Swinteck, N; Runge, K; Deymier-Black, A; Hoying, J B

2015-01-01

We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

Effect of HeNe laser on calcium signals in sperm cells

Science.gov (United States)

Lubart, Rachel; Friedmann, Harry; Cohen, Natalie; Brietbart, Haim

1998-12-01

Irradiation of mouse spermatozoa by 630 nm HeNe laser was found to enhance calcium transport in these cells. The change in Ca transport was investigated through two approaches, the first employing the fluorescent Ca indicator, Fluo-3 AM and a fluorescence microscopic system, and the second the radiolabeled Ca uptake. In both approaches the effect of light on Ca transport was abrogated in the absence of Ca during the irradiation time, indicating that the effect of light is Ca-dependent. The stimulatory effect of light on Ca uptake was inhibited by treatment with catalase, suggesting H2O2 to be involved in light stimulated Ca2+ uptake. The stimulatory effect of light on Ca uptake was abolished in the presence of a voltage-dependent Ca-channel inhibitor, nifedipine, indicating the involvement of a plasma membrane, voltage- dependent Ca-channel. In contrast, addition of nifedipine prior to the HeNe laser irradiation did not affect the light-induced rise in intracellular Ca levels, as measured with Fluo-3 loaded sperm cells. Therefore, it can be concluded that this Ca influx occurs via a voltage- insensitive Ca-channel. The stimulatory effect of light on Ca uptake was almost completely abolished by the mitochondrial uncoupler FCCP. These data imply that light affects the mitochondrial Ca transport mechanisms. It is well known that Ca influx from an extracellular environment is an essential component of a signaling cascade leading to fertilization.

Plasticity of calcium-permeable AMPA glutamate receptors in Pro-opiomelanocortin neurons.

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Suyama, Shigetomo; Ralevski, Alexandra; Liu, Zhong-Wu; Dietrich, Marcelo O; Yada, Toshihiko; Simonds, Stephanie E; Cowley, Michael A; Gao, Xiao-Bing; Diano, Sabrina; Horvath, Tamas L

2017-08-01

POMC neurons integrate metabolic signals from the periphery. Here, we show in mice that food deprivation induces a linear current-voltage relationship of AMPAR-mediated excitatory postsynaptic currents (EPSCs) in POMC neurons. Inhibition of EPSCs by IEM-1460, an antagonist of calcium-permeable (Cp) AMPARs, diminished EPSC amplitude in the fed but not in the fasted state, suggesting entry of GluR2 subunits into the AMPA receptor complex during food deprivation. Accordingly, removal of extracellular calcium from ACSF decreased the amplitude of mEPSCs in the fed but not the fasted state. Ten days of high-fat diet exposure, which was accompanied by elevated leptin levels and increased POMC neuronal activity, resulted in increased expression of Cp-AMPARs on POMC neurons. Altogether, our results show that entry of calcium via Cp-AMPARs is inherent to activation of POMC neurons, which may underlie a vulnerability of these neurons to calcium overload while activated in a sustained manner during over-nutrition.

Calcium: the molecular basis of calcium action in biology and medicine

National Research Council Canada - National Science Library

Pochet, Roland; Donato, Rosario

2000-01-01

... of Calcium Calcium Signalling in Excitable Cells Ca2+ Release in Muscle Cells by N. Macrez and J. Mironneau Calcium Signalling in Neurons Exemplified by Rat Sympathetic Ganglion Cells by S.J. M...

Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

Science.gov (United States)

Vatsa, Parul; Chiltz, Annick; Luini, Estelle; Vandelle, Elodie; Pugin, Alain; Roblin, Gabriel

2011-07-01

The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²⁺](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 μM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²⁺](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl₃, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²⁺](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

Science.gov (United States)

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Extracellular Ca²⁺ acts as a mediator of communication from neurons to glia.

Science.gov (United States)

Torres, Arnulfo; Wang, Fushun; Xu, Qiwu; Fujita, Takumi; Dobrowolski, Radoslaw; Willecke, Klaus; Takano, Takahiro; Nedergaard, Maiken

2012-01-24

Defining the pathways through which neurons and astrocytes communicate may contribute to the elucidation of higher central nervous system functions. We investigated the possibility that decreases in extracellular calcium ion concentration ([Ca(2+)](e)) that occur during synaptic transmission might mediate signaling from neurons to glia. Using noninvasive photolysis of the photolabile Ca(2+) buffer diazo-2 {N-[2-[2-[2-[bis(carboxymethyl)amino]-5-(diazoacetyl)phenoxy]ethoxy]-4-methylphenyl]-N-(carboxymethyl)-, tetrapotassium salt} to reduce [Ca(2+)](e) or caged glutamate to simulate glutamatergic transmission, we found that a local decline in extracellular Ca(2+) triggered astrocytic adenosine triphosphate (ATP) release and astrocytic Ca(2+) signaling. In turn, activation of purinergic P2Y1 receptors on a subset of inhibitory interneurons initiated the generation of action potentials by these interneurons, thereby enhancing synaptic inhibition. Thus, astrocytic ATP release evoked by an activity-associated decrease in [Ca(2+)](e) may provide a negative feedback mechanism that potentiates inhibitory transmission in response to local hyperexcitability.

Calcium Signalling in Plant Biotic Interactions

Directory of Open Access Journals (Sweden)

Didier Aldon

2018-02-01

Full Text Available Calcium (Ca2+ is a universal second messenger involved in various cellular processes, leading to plant development and to biotic and abiotic stress responses. Intracellular variation in free Ca2+ concentration is among the earliest events following the plant perception of environmental change. These Ca2+ variations differ in their spatio-temporal properties according to the nature, strength and duration of the stimulus. However, their conversion into biological responses requires Ca2+ sensors for decoding and relaying. The occurrence in plants of calmodulin (CaM but also of other sets of plant-specific Ca2+ sensors such as calmodulin-like proteins (CMLs, Ca2+-dependent protein kinases (CDPKs and calcineurin B-like proteins (CBLs indicate that plants possess specific tools and machineries to convert Ca2+ signals into appropriate responses. Here, we focus on recent progress made in monitoring the generation of Ca2+ signals at the whole plant or cell level and their long distance propagation during biotic interactions. The contribution of CaM/CMLs and CDPKs in plant immune responses mounted against bacteria, fungi, viruses and insects are also presented.

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

International Nuclear Information System (INIS)

Blanc-Brude, Olivier P.; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

2005-01-01

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR 1 ). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR 1 -deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR 1 -specific agonists and inhibitors were used to demonstrate that PAR 1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR 1 and not PAR 2 . These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis

Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

DEFF Research Database (Denmark)

Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

2002-01-01

that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

Calcium signals in olfactory neurons.

Science.gov (United States)

Tareilus, E; Noé, J; Breer, H

1995-11-09

Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

In vitro Determination of Extracellular Proteins from Xylella fastidiosa.

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Mendes, Juliano S; Santiago, André S; Toledo, Marcelo A S; Horta, Maria A C; de Souza, Alessandra A; Tasic, Ljubica; de Souza, Anete P

2016-01-01

The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa . Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa . Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3-30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.

Models of calcium signalling

CERN Document Server

Dupont, Geneviève; Kirk, Vivien; Sneyd, James

2016-01-01

This book discusses the ways in which mathematical, computational, and modelling methods can be used to help understand the dynamics of intracellular calcium. The concentration of free intracellular calcium is vital for controlling a wide range of cellular processes, and is thus of great physiological importance. However, because of the complex ways in which the calcium concentration varies, it is also of great mathematical interest.This book presents the general modelling theory as well as a large number of specific case examples, to show how mathematical modelling can interact with experimental approaches, in an interdisciplinary and multifaceted approach to the study of an important physiological control mechanism. Geneviève Dupont is FNRS Research Director at the Unit of Theoretical Chronobiology of the Université Libre de Bruxelles;Martin Falcke is head of the Mathematical Cell Physiology group at the Max Delbrück Center for Molecular Medicine, Berlin;Vivien Kirk is an Associate Professor in the Depar...

Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

DEFF Research Database (Denmark)

Kennedy, Arion; Martinez, Kristina; Chung, Soonkyu

2010-01-01

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor kappaB (NFkappaB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated...... that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated......, and suppression of peroxisome proliferator activated receptor gamma protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca2+]i levels, particularly calcium from the ER....

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

Science.gov (United States)

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I

2015-08-03

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

Association analysis between mitogen-activated protein/extracellular signal-regulated kinase (MEK) gene polymorphisms and depressive disorder in the Han Chinese population.

Science.gov (United States)

Hu, Yingyan; Hong, Wu; Smith, Alicia; Yu, Shunying; Li, Zezhi; Wang, Dongxiang; Yuan, Chengmei; Cao, Lan; Wu, Zhiguo; Huang, Jia; Fralick, Drew; Phillips, Michael Robert; Fang, Yiru

2017-11-01

Recent research findings suggest that BDNF and BDNF signaling pathways participate in the development of major depressive disorder. Mitogen-activated extracellular signal-regulated kinase (MEK) is the most important kinase in the extracellular signal-regulated kinase pathway, and the extracellular signal-regulated kinase pathway is the key signaling pathway of BDNF, so it may play a role in development of depressive disorder. The aim of this study is to investigate the association between polymorphisms of the MAP2K1 (also known as MEK) gene and depressive disorder. Three single nucleotide polymorphisms (SNPs), were significantly associated with depressive disorder: rs1549854 (p = 0.006), rs1432441 (p = 0.025), and rs7182853 (p = 0.039). When subdividing the sample by gender, two of the SNPs remained statistically associated with depressive disorder in females: rs1549854 (p = 0.013) and rs1432441 (p = 0.04). The rs1549854 and rs1432441 polymorphisms of the MAP2K1 gene may be associated with major depressive disorder, especially in females. This study is the first to report that the MAP2K1 gene may be a genetic marker for depressive disorder. Copyright © 2017 Elsevier B.V. All rights reserved.

TRPC1, STIM1, and ORAI influence signal-regulated intracellular and endoplasmic reticulum calcium dynamics in human myometrial cells.

Science.gov (United States)

Murtazina, Dilyara A; Chung, Daesuk; Ulloa, Aida; Bryan, Emily; Galan, Henry L; Sanborn, Barbara M

2011-08-01

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.

SMOC can act as both an antagonist and an expander of BMP signaling.

Science.gov (United States)

Thomas, J Terrig; Eric Dollins, D; Andrykovich, Kristin R; Chu, Tehyen; Stultz, Brian G; Hursh, Deborah A; Moos, Malcolm

2017-03-21

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone ( pent ), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

Directory of Open Access Journals (Sweden)

Nunzio Iraci

2016-02-01

Full Text Available Extracellular vesicles (EVs are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

Science.gov (United States)

Bkaily, Ghassan; El-Bizri, Nesrine; Bui, Michel; Sukarieh, Rami; Jacques, Danielle; Fu, Michael L X

2003-03-01

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.

Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule.

Science.gov (United States)

Forsyth, M H; Cover, T L

2000-06-01

Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of 100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5alpha produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori. Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi. We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.

Angiotensin effects on calcium and steroidogenesis in adrenal glomerulosa cells

International Nuclear Information System (INIS)

Elliott, M.E.; Siegel, F.L.; Hadjokas, N.E.; Goodfriend, T.L.

1985-01-01

We investigated the role of cellular calcium pools in angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Angiotensin II decreased the size of the exchangeable cell calcium pool by 34%, consistent with previous observations that angiotensin II causes decreased uptake of 45 Ca+2 into cells and increased efflux of 45 Ca+2 from preloaded cells. Atomic absorption spectroscopy showed that angiotension II caused a decrease of 21% in total cellular calcium. Angiotensin II caused efflux of 45 Ca+2 in the presence of EGTA and retarded uptake of 45 Ca+2 when choline was substituted for sodium, suggesting that hormone effects on calcium pools do not involve influx of trigger calcium or sodium. Cells incubated in calcium-free buffer and 0.1 mM or 0.5 mM EGTA synthesized reduced (but still significant) amounts of the steroid in response to hormone. Cells incubated in increasing concentrations of extracellular calcium contained increasing amounts of intracellular calcium and synthesized increasing amounts of aldosterone in response to angiotensin II. These results point to the participation of intracellular calcium pools in angiotensin II-stimulated steroidogenesis and the importance of extracellular calcium in maintaining these pools

Calcium-Dependent Protein Kinases in Phytohormone Signaling Pathways

Directory of Open Access Journals (Sweden)

Wuwu Xu

2017-11-01

Full Text Available Calcium-dependent protein kinases (CPKs/CDPKs are Ca2+-sensors that decode Ca2+ signals into specific physiological responses. Research has reported that CDPKs constitute a large multigene family in various plant species, and play diverse roles in plant growth, development, and stress responses. Although numerous CDPKs have been exhaustively studied, and many of them have been found to be involved in plant hormone biosynthesis and response mechanisms, a comprehensive overview of the manner in which CDPKs participate in phytohormone signaling pathways, regulating nearly all aspects of plant growth, has not yet been undertaken. In this article, we reviewed the structure of CDPKs and the mechanism of their subcellular localization. Some CDPKs were elucidated to influence the intracellular localization of their substrates. Since little work has been done on the interaction between CDPKs and cytokinin signaling pathways, or on newly defined phytohormones such as brassinosteroids, strigolactones and salicylic acid, this paper mainly focused on discussing the integral associations between CDPKs and five plant hormones: auxins, gibberellins, ethylene, jasmonates, and abscisic acid. A perspective on future work is provided at the end.

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

Science.gov (United States)

Schleede, Justin; Blair, Seth S

2015-10-01

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

Two-photon activation of endogenous store-operated calcium channels without optogenetics

Science.gov (United States)

Cheng, Pan; Tang, Wanyi; He, Hao

2018-02-01

Store-operated calcium (SOC) channels, regulated by intracellular Ca2+ store, are the essential pathway of calcium signaling and participate in a wide variety of cellular activities such as gene expression, secretion and immune response1. However, our understanding and regulation of SOC channels are mainly based on pharmacological methods. Considering the unique advantages of optical control, optogenetic control of SOC channels has been developed2. However, the process of genetic engineering to express exogenous light-sensitive protein is complicated, which arouses concerns about ethic difficulties in some research of animal and applications in human. In this report, we demonstrate rapid, robust and reproducible two-photon activation of endogenous SOC channels by femtosecond laser without optogenetics. We present that the short-duration two-photon scanning on subcellular microregion induces slow Ca2+ influx from extracellular medium, which can be eliminated by removing extracellular Ca2+. Block of SOC channels using various pharmacological inhibitors or knockdown of SOC channels by RNA interference reduce the probability of two-photon activated Ca2+ influx. On the contrary, overexpression of SOC channels can increase the probability of Ca2+ influx by two-photon scanning. These results collectively indicate Ca2+ influx through two-photon activated SOC channels. Different from classical pathway of SOC entry activated by Ca2+ store depletion, STIM1, the sensor protein of Ca2+ level in endoplasmic reticulum, does not show any aggregation or migration in this two-photon activated Ca2+ influx, which rules out the possibility of intracellular Ca2+ store depletion. Thereby, we propose this all-optical method of two-photon activation of SOC channels is of great potential to be widely applied in the research of cell calcium signaling and related biological research.

Wound healing, calcium signaling, and other novel pathways are associated with the formation of butterfly eyespots.

Science.gov (United States)

Özsu, Nesibe; Monteiro, Antónia

2017-10-16

One hypothesis surrounding the origin of novel traits is that they originate from the co-option of pre-existing genes or larger gene regulatory networks into novel developmental contexts. Insights into a trait's evolutionary origins can, thus, be gained via identification of the genes underlying trait development, and exploring whether those genes also function in other developmental contexts. Here we investigate the set of genes associated with the development of eyespot color patterns, a trait that originated once within the Nymphalid family of butterflies. Although several genes associated with eyespot development have been identified, the eyespot gene regulatory network remains largely unknown. In this study, next-generation sequencing and transcriptome analyses were used to identify a large set of genes associated with eyespot development of Bicyclus anynana butterflies, at 3-6 h after pupation, prior to the differentiation of the color rings. Eyespot-associated genes were identified by comparing the transcriptomes of homologous micro-dissected wing tissues that either develop or do not develop eyespots in wild-type and a mutant line of butterflies, Spotty, with extra eyespots. Overall, 186 genes were significantly up and down-regulated in wing tissues that develop eyespots compared to wing tissues that do not. Many of the differentially expressed genes have yet to be annotated. New signaling pathways, including the Toll, Fibroblast Growth Factor (FGF), extracellular signal-regulated kinase (ERK) and/or Jun N-terminal kinase (JNK) signaling pathways are associated for the first time with eyespot development. In addition, several genes involved in wound healing and calcium signaling were also found to be associated with eyespots. Overall, this study provides the identity of many new genes and signaling pathways associated with eyespots, and suggests that the ancient wound healing gene regulatory network may have been co-opted to cells at the center of the

The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Liu Yukan; Lu Qiaozhi; Ji Huijiao; Zhao Xiaoli; Zhang Ming [College of Life Science, ZheJiang University, Hangzhou 310058 (China); Pei Rui [XiJing Hospital, Fourth Military Medical University, Xi An 710032 (China); Zhou Guoshun [Huzhou Central Hospital, Huzhou, Zhejiang Province 313000 (China); Tang, Rui Kang, E-mail: Lyk_cn@yahoo.com.c [Department of Chemistry, College of Science, ZheJiang University, Hangzhou 310027 (China)

2009-04-15

The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca{sup 2+}) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca{sup 2+} and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca{sup 2+} and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and alpha-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca{sup 2+} concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca{sup 2+} concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca{sup 2+} and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca{sup 2+} and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.

Cytoplasmic organelles determine complexity and specificity of calcium signalling in adrenal chromaffin cells

Czech Academy of Sciences Publication Activity Database

Garsia-Sancho, J.; Verkhratsky, Alexei

2008-01-01

Roč. 192, č. 2 (2008), s. 263-271 ISSN 1748-1708 Institutional research plan: CEZ:AV0Z50390512 Keywords : Ca2+ signalling * calcium microdomains * chromaffin cells Subject RIV: JE - Non-nuclear Energetics, Energy Consumption ; Use Impact factor: 2.455, year: 2008

Congenital heart block maternal sera autoantibodies target an extracellular epitope on the α1G T-type calcium channel in human fetal hearts.

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Linn S Strandberg

Full Text Available Congenital heart block (CHB is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB.We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation. Using human fetal hearts (20-22 wks gestation, our immunoprecipitation (IP, Western blot analysis and immunofluorescence (IF staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I. Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN cells.Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

Science.gov (United States)

Sergé, Arnauld; Bernard, Anne-Marie; Phélipot, Marie-Claire; Bertaux, Nicolas; Fallet, Mathieu; Grenot, Pierre; Marguet, Didier; He, Hai-Tao; Hamon, Yannick

2013-01-01

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells. PMID:24086124

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

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Justin Schleede

2015-10-01

Full Text Available The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog. However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

The Role of nAChR and Calcium Signaling in Pancreatic Cancer Initiation and Progression

Energy Technology Data Exchange (ETDEWEB)

Schaal, Courtney [Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 (United States); Padmanabhan, Jaya [Department of Molecular Medicine and USF Health Byrd Alzheimer’s Institute, University of South Florida, 4001 E. Fletcher Ave., Tampa, FL 33612 (United States); Chellappan, Srikumar, E-mail: Srikumar.Chellappan@moffitt.org [Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 (United States)

2015-07-31

Pancreatic cancer shows a strong correlation with smoking and the current therapeutic strategies have been relatively ineffective in improving the survival of patients. Efforts have been made over the past many years to understand the molecular events that drive the initiation and progression of pancreatic cancer, especially in the context of smoking. It has become clear that components of tobacco smoke not only initiate these cancers, especially pancreatic ductal adenocarcinomas (PDACs) through their mutagenic properties, but can also promote the growth and metastasis of these tumors by stimulating cell proliferation, angiogenesis, invasion and epithelial-mesenchymal transition. Studies in cell culture systems, animal models and human samples have shown that nicotinic acetylcholine receptor (nAChR) activation enhances these tumor-promoting events by channeling signaling through multiple pathways. In this context, signaling through calcium channels appear to facilitate pancreatic cancer growth by itself or downstream of nAChRs. This review article highlights the role of nAChR downstream signaling events and calcium signaling in the growth, metastasis as well as drug resistance of pancreatic cancer.

The Role of nAChR and Calcium Signaling in Pancreatic Cancer Initiation and Progression

International Nuclear Information System (INIS)

Schaal, Courtney; Padmanabhan, Jaya; Chellappan, Srikumar

2015-01-01

Pancreatic cancer shows a strong correlation with smoking and the current therapeutic strategies have been relatively ineffective in improving the survival of patients. Efforts have been made over the past many years to understand the molecular events that drive the initiation and progression of pancreatic cancer, especially in the context of smoking. It has become clear that components of tobacco smoke not only initiate these cancers, especially pancreatic ductal adenocarcinomas (PDACs) through their mutagenic properties, but can also promote the growth and metastasis of these tumors by stimulating cell proliferation, angiogenesis, invasion and epithelial-mesenchymal transition. Studies in cell culture systems, animal models and human samples have shown that nicotinic acetylcholine receptor (nAChR) activation enhances these tumor-promoting events by channeling signaling through multiple pathways. In this context, signaling through calcium channels appear to facilitate pancreatic cancer growth by itself or downstream of nAChRs. This review article highlights the role of nAChR downstream signaling events and calcium signaling in the growth, metastasis as well as drug resistance of pancreatic cancer

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

Science.gov (United States)

Qin, S; Chock, P B

2001-07-10

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for

Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate

Science.gov (United States)

Molina, Anthony J A; Verzi, Michael P; Birnbaum, Andrea D; Yamoah, Ebenezer N; Hammar, Katherine; Smith, Peter J S; Malchow, Robert Paul

2004-01-01

Self-referencing H+-selective microelectrodes were used to measure extracellular H+ fluxes from horizontal cells isolated from the skate retina. A standing H+ flux was detected from quiescent cells, indicating a higher concentration of free hydrogen ions near the extracellular surface of the cell as compared to the surrounding solution. The standing H+ flux was reduced by removal of extracellular sodium or application of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), suggesting activity of a Na+–H+ exchanger. Glutamate decreased H+ flux, lowering the concentration of free hydrogen ions around the cell. AMPA/kainate receptor agonists mimicked the response, and the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) eliminated the effects of glutamate and kainate. Metabotropic glutamate agonists were without effect. Glutamate-induced alterations in H+ flux required extracellular calcium, and were abolished when cells were bathed in an alkaline Ringer solution. Increasing intracellular calcium by photolysis of the caged calcium compound NP-EGTA also altered extracellular H+ flux. Immunocytochemical localization of the plasmalemma Ca2+–H+-ATPase (PMCA pump) revealed intense labelling within the outer plexiform layer and on isolated horizontal cells. Our results suggest that glutamate modulation of H+ flux arises from calcium entry into cells with subsequent activation of the plasmalemma Ca2+–H+-ATPase. These neurotransmitter-induced changes in extracellular pH have the potential to play a modulatory role in synaptic processing in the outer retina. However, our findings argue against the hypothesis that hydrogen ions released by horizontal cells normally act as the inhibitory feedback neurotransmitter onto photoreceptor synaptic terminals to create the surround portion of the centre-surround receptive fields of retinal neurones. PMID:15272044

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

Science.gov (United States)

Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

2006-08-15

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

The matricellular receptor LRP1 forms an interface for signaling and endocytosis in modulation of the extracellular tumor environment

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Bart eVan Gool

2015-11-01

Full Text Available The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1 has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease-inhibitor complexes and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents.This mini-review focuses on LRP1’s role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

International Nuclear Information System (INIS)

Phair, R.D.; Hai, C.M.

1986-01-01

A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

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Chen-Shuang Li

2016-01-01

Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

Sparks, signals and shock absorbers: how dystrophin loss causes muscular dystrophy.

Science.gov (United States)

Batchelor, Clare L; Winder, Steve J

2006-04-01

The dystrophin-glycoprotein complex (DGC) can be considered as a specialized adhesion complex, linking the extracellular matrix to the actin cytoskeleton, primarily in muscle cells. Mutations in several components of the DGC lead to its partial or total loss, resulting in various forms of muscular dystrophy. These typically manifest as progressive wasting diseases with loss of muscle integrity. Debate is ongoing about the precise function of the DGC: initially a strictly mechanical role was proposed but it has been suggested that there is aberrant calcium handling in muscular dystrophy and, more recently, changes in MAP kinase and GTPase signalling have been implicated in the aetiology of the disease. Here, we discuss new and interesting developments in these aspects of DGC function and attempt to rationalize the mechanical, calcium and signalling hypotheses to provide a unifying hypothesis of the underlying process of muscular dystrophy.

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

International Nuclear Information System (INIS)

O’Day, Danton H.; Huber, Robert J.; Suarez, Andres

2012-01-01

Highlights: ► Extracellular calmodulin is present throughout growth and development in Dictyostelium. ► Extracellular calmodulin localizes within the ECM during development. ► Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. ► Extracellular calmodulin exists in eukaryotic microbes. ► Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca 2+ /CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

Effect of TGFβ on calcium signaling in megakaryocytes

Energy Technology Data Exchange (ETDEWEB)

Yan, Jing [Department of Physiology I, University of Tübingen, Tübingen (Germany); Schmid, Evi [Department of Physiology I, University of Tübingen, Tübingen (Germany); Department of Pediatric Surgery and Pediatric Urology, University Children' s Hospital Tübingen, Tübingen (Germany); Almilaji, Ahmad; Shumilina, Ekaterina [Department of Physiology I, University of Tübingen, Tübingen (Germany); Borst, Oliver [Department of Physiology I, University of Tübingen, Tübingen (Germany); Department of Cardiology & Cardiovascular Medicine, University of Tübingen, Tübingen (Germany); Laufer, Stefan [Department of Pharmacy, University of Tübingen, Tübingen (Germany); Gawaz, Meinrad [Department of Cardiology & Cardiovascular Medicine, University of Tübingen, Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology I, University of Tübingen, Tübingen (Germany)

2015-05-22

TGFβ is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFβ may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/β), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca{sup 2+} entry (SOCE). The present study explored whether TGFβ influences Ca{sup 2+} signaling in megakaryocytes. [Ca{sup 2+}]{sub i} was determined by Fura-2 fluorescence and SOCE from the increase of [Ca{sup 2+}]{sub i} following re-addition of extracellular Ca{sup 2+} after store depletion by removal of extracellular Ca{sup 2+} and inhibition of the sarcoendoplasmatic Ca{sup 2+} ATPase (SERCA) with thapsigargin (1 μM). As a result, TGFβ (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) and NFκB inhibitor wogonin (100 μM). In conclusion, TGFβ is a powerful regulator of store operated Ca{sup 2+} entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB. - Highlights: • TGFβ up-regulates store operated Ca{sup 2+} entry (SOCE) in megakaryocytes. • The effect of TGFβ on SOCE is blunted by p38 kinase inhibitor Skepinone-L. • The effect of TGFβ on SOCE is virtually abrogated by SGK1 inhibitor EMD638683. • The effect of TGFβ on SOCE is almost abolished by NFκB inhibitor wogonin. • The effect of TGFβ is expected to enhance sensitivity of platelets to activation.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

Science.gov (United States)

Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

2017-10-01

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Smad signaling pathway in pathogenesis of kidney injury induced by calcium oxalate stone in rats

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Fan Zhang

2016-10-01

Full Text Available Objective: To investigate the involvement of Smad signaling pathway in the pathogenesis of kidney injury induced by calcium oxalate stone in rats to provide a reference for clinical treatment. Methods: Clean SD rats were randomly divided into 3 group, namely the control group, model group and pirfenidone group. Ethylene glycol + αhydroxy vitamin D3 was used as a stone-inducing agent to replicate the renal calcium oxalate stone model. Rats in the pirfenidone group were treated with pirfenidone intragastric administration. The serum Cr, BUN and 24-hour oxalate and calcium in renal tissues were assayed. The expressions of Bax/ Bcl2 protein, Caspase3 protein, TGFβ, Smad1, Smad2 and Smad3 proteins were detected by the fluorescent quantitation PCR method. Results: Compared with the rats of the control group, the results showed that the levels of serum BUN, Cr and 24-hour oxalate in rats of the model group were increased greatly, Bax and Caspase3 mRNA also increased while the level of Bcl2 decreased significantly, and the expressions of TGFβ, Smad1, Smad2 and Smad3 proteins increased distinctly as well (P<0.01. These abnormal parameters could be normalized effectively by pirfenidone. Conclusions: Activated TGFβ/Smad signaling pathway is involved in the pathogenesis of kidney injury induced by calcium oxalate stone in rats.

An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells

International Nuclear Information System (INIS)

Borle, A.B.

1990-01-01

An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total call calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca 2+ compartmentalization, but the methods suffer from the possibility of Ca 2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45 Ca uptake or desaturation curves have been used to study the distribution of Ca 2+ among various kinetic pools in living cells and their rate of Ca 2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45 Ca uptake can detect instantaneous changes in calcium influx, while 45 Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles

Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

International Nuclear Information System (INIS)

Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

2009-01-01

Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects.

LENUS (Irish Health Repository)

Lyng, F M

2006-04-01

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of

Atorvastatin calcium inhibits phenotypic modulation of PDGF-BB-induced VSMCs via down-regulation the Akt signaling pathway.

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Chen, Shuang; Liu, Baoqin; Kong, Dehui; Li, Si; Li, Chao; Wang, Huaqin; Sun, Yingxian

2015-01-01

Plasticity of vascular smooth muscle cells (VSMCs) plays a central role in the onset and progression of proliferative vascular diseases. In adult tissue, VSMCs exist in a physiological contractile-quiescent phenotype, which is defined by lack of the ability of proliferation and migration, while high expression of contractile marker proteins. After injury to the vessel, VSMC shifts from a contractile phenotype to a pathological synthetic phenotype, associated with increased proliferation, migration and matrix secretion. It has been demonstrated that PDGF-BB is a critical mediator of VSMCs phenotypic switch. Atorvastatin calcium, a selective inhibitor of 3-hydroxy-3-methyl-glutaryl l coenzyme A (HMG-CoA) reductase, exhibits various protective effects against VSMCs. In this study, we investigated the effects of atorvastatin calcium on phenotype modulation of PDGF-BB-induced VSMCs and the related intracellular signal transduction pathways. Treatment of VSMCs with atorvastatin calcium showed dose-dependent inhibition of PDGF-BB-induced proliferation. Atorvastatin calcium co-treatment inhibited the phenotype modulation and cytoskeleton rearrangements and improved the expression of contractile phenotype marker proteins such as α-SM actin, SM22α and calponin in comparison with PDGF-BB alone stimulated VSMCs. Although Akt phosphorylation was strongly elicited by PDGF-BB, Akt activation was attenuated when PDGF-BB was co-administrated with atorvastatin calcium. In conclusion, atorvastatin calcium inhibits phenotype modulation of PDGF-BB-induced VSMCs and activation of the Akt signaling pathway, indicating that Akt might play a vital role in the modulation of phenotype.

Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR are associated with increased ER to cytosol calcium gradient.

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Marianna Ranieri

Full Text Available In humans, gain-of-function mutations of the calcium-sensing receptor (CASR gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA and reducing expression of Plasma Membrane Calcium-ATPase (PMCA. Wild-type CaSR (hCaSR-wt and its gain-of-function (hCaSR-R990G; hCaSR-N124K variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate receptor inputs to cell function.

Dynamical patterns of calcium signaling in a functional model of neuron-astrocyte networks

DEFF Research Database (Denmark)

Postnov, D.E.; Koreshkov, R.N.; Brazhe, N.A.

2009-01-01

We propose a functional mathematical model for neuron-astrocyte networks. The model incorporates elements of the tripartite synapse and the spatial branching structure of coupled astrocytes. We consider glutamate-induced calcium signaling as a specific mode of excitability and transmission...... in astrocytic-neuronal networks. We reproduce local and global dynamical patterns observed experimentally....

Signaling domain of Sonic Hedgehog as cannibalistic calcium-regulated zinc-peptidase.

Directory of Open Access Journals (Sweden)

Rocio Rebollido-Rios

2014-07-01

Full Text Available Sonic Hedgehog (Shh is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog, of which all members harbor a structurally well-defined Zn2+ center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double-Ca2+ center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1 a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2 a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on pH.

Movement of calcium signals and calcium-binding proteins: firewalls, traps and tunnels.

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Barrow, S L; Sherwood, M W; Dolman, N J; Gerasimenko, O V; Voronina, S G; Tepikin, A V

2006-06-01

In the board game 'Snakes and Ladders', placed on the image of a pancreatic acinar cell, calcium ions have to move from release sites in the secretory region to the nucleus. There is another important contraflow - from calcium entry channels in the basal part of the cell to ER (endoplasmic reticulum) terminals in the secretory granule region. Both transport routes are perilous as the messenger can disappear in any place on the game board. It can be grabbed by calcium ATPases of the ER (masquerading as a snake but functioning like a ladder) and tunnelled through its low buffering environment, it can be lured into the whirlpools of mitochondria uniporters and forced to regulate the tricarboxylic acid cycle, and it can be permanently placed inside the matrix of secretory granules and released only outside the cell. The organelles could trade calcium (e.g. from the ER to mitochondria and vice versa) almost depriving this ion the light of the cytosol and noble company of cytosolic calcium buffers. Altogether it is a rich and colourful story.

Multiparameter imaging of calcium and abscisic acid and high-resolution quantitative calcium measurements using R-GECO1-mTurquoise in Arabidopsis.

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Waadt, Rainer; Krebs, Melanie; Kudla, Jörg; Schumacher, Karin

2017-10-01

Calcium signals occur in specific spatio-temporal patterns in response to various stimuli and are coordinated with, for example, hormonal signals, for physiological and developmental adaptations. Quantification of calcium together with other signalling molecules is required for correlative analyses and to decipher downstream calcium-decoding mechanisms. Simultaneous in vivo imaging of calcium and abscisic acid has been performed here to investigate the interdependence of the respective signalling processes in Arabidopsis thaliana roots. Advanced ratiometric genetically encoded calcium indicators have been generated and in vivo calcium calibration protocols were established to determine absolute calcium concentration changes in response to auxin and ATP. In roots, abscisic acid induced long-term basal calcium concentration increases, while auxin triggered rapid signals in the elongation zone. The advanced ratiometric calcium indicator R-GECO1-mTurquoise exhibited an increased calcium signal resolution compared to commonly used Förster resonance energy transfer-based indicators. Quantitative calcium measurements in Arabidopsis root tips using R-GECO1-mTurquoise revealed detailed maps of absolute calcium concentration changes in response to auxin and ATP. Calcium calibration protocols using R-GECO1-mTurquoise enabled high-resolution quantitative imaging of resting cytosolic calcium concentrations and their dynamic changes that revealed distinct hormonal and ATP responses in roots. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

International Nuclear Information System (INIS)

Chubinskiy-Nadezhdin, Vladislav I.; Vasileva, Valeria Y.; Pugovkina, Natalia A.; Vassilieva, Irina O.; Morachevskaya, Elena A.; Nikolsky, Nikolay N.; Negulyaev, Yuri A.

2017-01-01

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances in hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca 2+ entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca 2+ -sensitive BK and SK channels was shown. • Local Ca 2+ influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca 2+ ] i . • Functional clustering of SACs and BK channels in stem cell membrane is proposed.

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

International Nuclear Information System (INIS)

Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

1989-01-01

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release

Calcium-Induced calcium release during action potential firing in developing inner hair cells.

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Iosub, Radu; Avitabile, Daniele; Grant, Lisa; Tsaneva-Atanasova, Krasimira; Kennedy, Helen J

2015-03-10

In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights

Calcium microdomains near R-type calcium channels control the induction of presynaptic LTP at parallel fiber to Purkinje cell synapses

Science.gov (United States)

Myoga, Michael H.; Regehr, Wade G.

2011-01-01

R-type calcium channels in postsynaptic spines signal through functional calcium microdomains to regulate a calcium-calmodulin sensitive potassium channel that in turn regulates postsynaptic hippocampal LTP. Here we ask whether R-type calcium channels in presynaptic terminals also signal through calcium microdomains to control presynaptic LTP. We focus on presynaptic LTP at parallel fiber to Purkinje cell synapses in the cerebellum (PF-LTP), which is mediated by calcium/calmodulin-stimulated adenylyl cyclases. Although most presynaptic calcium influx is through N-type and P/Q-type calcium channels, blocking these channels does not disrupt PF-LTP, but blocking R-type calcium channels does. Moreover, global calcium signaling cannot account for the calcium dependence of PF-LTP because R-type channels contribute modestly to overall calcium entry. These findings indicate that within presynaptic terminals, R-type calcium channels produce calcium microdomains that evoke presynaptic LTP at moderate frequencies that do not greatly increase global calcium levels,. PMID:21471358

Store-Operated Calcium Entries Control Neural Stem Cell Self-Renewal in the Adult Brain Subventricular Zone.

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Domenichini, Florence; Terrié, Elodie; Arnault, Patricia; Harnois, Thomas; Magaud, Christophe; Bois, Patrick; Constantin, Bruno; Coronas, Valérie

2018-05-01

The subventricular zone (SVZ) is the major stem cell niche in the brain of adult mammals. Within this region, neural stem cells (NSC) proliferate, self-renew and give birth to neurons and glial cells. Previous studies underlined enrichment in calcium signaling-related transcripts in adult NSC. Because of their ability to mobilize sustained calcium influxes in response to a wide range of extracellular factors, store-operated channels (SOC) appear to be, among calcium channels, relevant candidates to induce calcium signaling in NSC whose cellular activities are continuously adapted to physiological signals from the microenvironment. By Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western blotting and immunocytochemistry experiments, we demonstrate that SVZ cells express molecular actors known to build up SOC, namely transient receptor potential canonical 1 (TRPC1) and Orai1, as well as their activator stromal interaction molecule 1 (STIM1). Calcium imaging reveals that SVZ cells display store-operated calcium entries. Pharmacological blockade of SOC with SKF-96365 or YM-58483 (also called BTP2) decreases proliferation, impairs self-renewal by shifting the type of SVZ stem cell division from symmetric proliferative to asymmetric, thereby reducing the stem cell population. Brain section immunostainings show that TRPC1, Orai1, and STIM1 are expressed in vivo, in SOX2-positive SVZ NSC. Injection of SKF-96365 in brain lateral ventricle diminishes SVZ cell proliferation and reduces the ability of SVZ cells to form neurospheres in vitro. The present study combining in vitro and in vivo approaches uncovers a major role for SOC in the control of SVZ NSC population and opens new fields of investigation for stem cell biology in health and disease. Stem Cells 2018;36:761-774. © AlphaMed Press 2018.

Neuronal Calcium Signaling in Metabolic Regulation and Adaptation to Nutrient Stress.

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Jayakumar, Siddharth; Hasan, Gaiti

2018-01-01

All organisms can respond physiologically and behaviorally to environmental fluxes in nutrient levels. Different nutrient sensing pathways exist for specific metabolites, and their inputs ultimately define appropriate nutrient uptake and metabolic homeostasis. Nutrient sensing mechanisms at the cellular level require pathways such as insulin and target of rapamycin (TOR) signaling that integrates information from different organ systems like the fat body and the gut. Such integration is essential for coordinating growth with development. Here we review the role of a newly identified set of integrative interneurons and the role of intracellular calcium signaling within these neurons, in regulating nutrient sensing under conditions of nutrient stress. A comparison of the identified Drosophila circuit and cellular mechanisms employed in this circuit, with vertebrate systems, suggests that the identified cell signaling mechanisms may be conserved for neural circuit function related to nutrient sensing by central neurons. The ideas proposed are potentially relevant for understanding the molecular basis of metabolic disorders, because these are frequently linked to nutritional stress.

Phenotypic variability in unicellular organisms: from calcium signalling to social behaviour.

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Vogel, David; Nicolis, Stamatios C; Perez-Escudero, Alfonso; Nanjundiah, Vidyanand; Sumpter, David J T; Dussutour, Audrey

2015-11-22

Historically, research has focused on the mean and often neglected the variance. However, variability in nature is observable at all scales: among cells within an individual, among individuals within a population and among populations within a species. A fundamental quest in biology now is to find the mechanisms that underlie variability. Here, we investigated behavioural variability in a unique unicellular organism, Physarum polycephalum. We combined experiments and models to show that variability in cell signalling contributes to major differences in behaviour underpinning some aspects of social interactions. First, following thousands of cells under various contexts, we identified distinct behavioural phenotypes: 'slow-regular-social', 'fast-regular-social' and 'fast-irregular-asocial'. Second, coupling chemical analysis and behavioural assays we found that calcium signalling is responsible for these behavioural phenotypes. Finally, we show that differences in signalling and behaviour led to alternative social strategies. Our results have considerable implications for our understanding of the emergence of variability in living organisms. © 2015 The Author(s).

Extracellular pH regulates zinc signaling via an Asp residue of the zinc-sensing receptor (ZnR/GPR39).

Science.gov (United States)

Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

2012-09-28

Zinc activates a specific Zn(2+)-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca(2+) responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na(+)/H(+) exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn(2+) binding site, His(17) or His(19), or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp(313) with alanine resulted in similar Ca(2+) responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na(+)/H(+) exchange at pH 7.4 and pH 6.5. Substitution of Asp(313) to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp(313), which was shown to modulate Zn(2+) binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.

Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)*

Science.gov (United States)

Cohen, Limor; Asraf, Hila; Sekler, Israel; Hershfinkel, Michal

2012-01-01

Zinc activates a specific Zn2+-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca2+ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na+/H+ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn2+ binding site, His17 or His19, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp313 with alanine resulted in similar Ca2+ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na+/H+ exchange at pH 7.4 and pH 6.5. Substitution of Asp313 to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp313, which was shown to modulate Zn2+ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity. PMID:22879599

Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

International Nuclear Information System (INIS)

Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

2011-01-01

Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

Role of Extracellular RNA and TLR3‐Trif Signaling in Myocardial Ischemia–Reperfusion Injury

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Chen, Chan; Feng, Yan; Zou, Lin; Wang, Larry; Chen, Howard H.; Cai, Jia‐Yan; Xu, Jun‐Mei; Sosnovik, David E.; Chao, Wei

2014-01-01

Background Toll‐like receptor 3 (TLR3) was originally identified as the receptor for viral RNA and represents a major host antiviral defense mechanism. TLR3 may also recognize extracellular RNA (exRNA) released from injured tissues under certain stress conditions. However, a role for exRNA and TLR3 in the pathogenesis of myocardial ischemic injury has not been tested. This study examined the role of exRNA and TLR3 signaling in myocardial infarction (MI), apoptosis, inflammation, and cardiac dysfunction during ischemia‐reperfusion (I/R) injury. Methods and Results Wild‐type (WT), TLR3−/−, Trif−/−, and interferon (IFN) α/β receptor‐1 deficient (IFNAR1−/−) mice were subjected to 45 minutes of coronary artery occlusion and 24 hours of reperfusion. Compared with WT, TLR3−/− or Trif−/− mice had smaller MI and better preserved cardiac function. Surprisingly, unlike TLR(2/4)‐MyD88 signaling, lack of TLR3‐Trif signaling had no impact on myocardial cytokines or neutrophil recruitment after I/R, but myocardial apoptosis was significantly attenuated in Trif−/− mice. Deletion of the downstream IFNAR1 had no effect on infarct size. Importantly, hypoxia and I/R led to release of RNA including microRNA from injured cardiomyocytes and ischemic heart, respectively. Necrotic cardiomyocytes induced a robust and dose‐dependent cytokine response in cultured cardiomyocytes, which was markedly reduced by RNase but not DNase, and partially blocked in TLR3‐deficient cardiomyocytes. In vivo, RNase administration reduced serum RNA level, attenuated myocardial cytokine production, leukocytes infiltration and apoptosis, and conferred cardiac protection against I/R injury. Conclusion TLR3‐Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R may contribute to myocardial inflammation and infarction. PMID:24390148

Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata – Tropiduridae strengthen classification in lizard evolution

Directory of Open Access Journals (Sweden)

Garcia Célia RS

2007-08-01

Full Text Available Abstract Background We have previously reported that a Teiid lizard red blood cells (RBCs such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition. Results Here we report, that RBCs from the tropidurid lizard Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like. Conclusion T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs, for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracelullar medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs.

Calcium Supplement Derived from Gallus gallus domesticus Promotes BMP-2/RUNX2/SMAD5 and Suppresses TRAP/RANK Expression through MAPK Signaling Activation

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Han Seok Yoo

2017-05-01

Full Text Available The present study evaluated the effects of a calcium (Ca supplement derived from Gallus gallus domesticus (GD on breaking force, microarchitecture, osteogenic differentiation and osteoclast differentiation factor expression in vivo in Ca-deficient ovariectomized (OVX rats. One percent of Ca supplement significantly improved Ca content and bone strength of the tibia. In micro-computed tomography analysis, 1% Ca supplement attenuated OVX- and low Ca-associated changes in bone mineral density, trabecular thickness, spacing and number. Moreover, 1% Ca-supplemented diet increased the expression of osteoblast differentiation marker genes, such as bone morphogenetic protein-2, Wnt3a, small mothers against decapentaplegic 1/5/8, runt-related transcription factor 2, osteocalcin and collagenase-1, while it decreased the expression of osteoclast differentiation genes, such as thrombospondin-related anonymous protein, cathepsin K and receptor activator of nuclear factor kappa B. Furthermore, 1% Ca-supplemented diet increased the levels of phosphorylated extracellular signal-regulated kinase and c-Jun N-terminal kinase. The increased expression of osteoblast differentiation marker genes and activation of mitogen-activated protein kinase signaling were associated with significant increases in trabecular bone volume, which plays an important role in the overall skeletal strength. Our results demonstrated that 1% Ca supplement inhibited osteoclastogenesis, stimulated osteoblastogenesis and restored bone loss in OVX rats.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

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Samigullin, Dmitry; Fatikhov, Nijaz; Khaziev, Eduard; Skorinkin, Andrey; Nikolsky, Eugeny; Bukharaeva, Ellya

2014-01-01

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers-which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal-has hitherto been technically impossible. With the aim of quantifying both Ca(2+) currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Herpes simplex virus triggers activation of calcium-signaling pathways

Science.gov (United States)

Cheshenko, Natalia; Del Rosario, Brian; Woda, Craig; Marcellino, Daniel; Satlin, Lisa M.; Herold, Betsy C.

2003-01-01

The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)–sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy. PMID:14568989

Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity.

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Ozcan, Lale; Wong, Catherine C L; Li, Gang; Xu, Tao; Pajvani, Utpal; Park, Sung Kyu Robin; Wronska, Anetta; Chen, Bi-Xing; Marks, Andrew R; Fukamizu, Akiyoshi; Backs, Johannes; Singer, Harold A; Yates, John R; Accili, Domenico; Tabas, Ira

2012-05-02

Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here, we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary hepatocytes and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis. Copyright © 2012 Elsevier Inc. All rights reserved.

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

International Nuclear Information System (INIS)

Lombardi, M.L.; Knecht, D.A.; Lee, J.

2008-01-01

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca 2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca 2+ ] i ) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca 2+ ] i . We observed small, brief, Ca 2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd 3+ ) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca 2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells

Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

Science.gov (United States)

Figueroa, Vania A.; Retamal, Mauricio A.; Cea, Luis A.; Salas, José D.; Vargas, Aníbal A.; Verdugo, Christian A.; Jara, Oscar; Martínez, Agustín D.; Sáez, Juan C.

2014-01-01

Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects. PMID:25237294

Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling.

Science.gov (United States)

Riethmüller, Michaela; Burger, Nils; Bauer, Georg

2015-12-01

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Regulation of PDGFC signalling and extracellular matrix composition by FREM1 in mice

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Fenny Wiradjaja

2013-11-01

Fras1-related extracellular matrix protein 1 (FREM1 is required for epidermal adhesion during embryogenesis, and mice lacking the gene develop fetal skin blisters and a range of other developmental defects. Mutations in members of the FRAS/FREM gene family cause diseases of the Fraser syndrome spectrum. Embryonic epidermal blistering is also observed in mice lacking PdgfC and its receptor, PDGFRα. In this article, we show that FREM1 binds to PDGFC and that this interaction regulates signalling downstream of PDGFRα. Fibroblasts from Frem1-mutant mice respond to PDGFC stimulation, but with a shorter duration and amplitude than do wild-type cells. Significantly, PDGFC-stimulated expression of the metalloproteinase inhibitor Timp1 is reduced in cells with Frem1 mutations, leading to reduced basement membrane collagen I deposition. These results show that the physical interaction of FREM1 with PDGFC can regulate remodelling of the extracellular matrix downstream of PDGFRα. We propose that loss of FREM1 function promotes epidermal blistering in Fraser syndrome as a consequence of reduced PDGFC activity, in addition to its stabilising role in the basement membrane.

Calcium signals in planetary embryos

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Morbidelli, Alessandro

2018-03-01

The calcium-isotope composition of planetary bodies in the inner Solar System correlates with the masses of such objects. This finding could have implications for our understanding of how the Solar System formed.

The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

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Charles Godbout

Full Text Available Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.

Calcium sensing in exocytosis

DEFF Research Database (Denmark)

Gustavsson, Natalia; Wu, Bingbing; Han, Weiping

2012-01-01

an increase in intracellular calcium levels. Besides the triggering role, calcium signaling modulates the precise amount and kinetics of vesicle release. Thus, it is a central question to understand the molecular machineries responsible for calcium sensing in exocytosis. Here we provide an overview of our...... current understanding of calcium sensing in neurotransmitter release and hormone secretion....

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

Directory of Open Access Journals (Sweden)

Dmitry eSamigullin

2015-01-01

Full Text Available At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 рА and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 µM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Optimization of culture media for extracellular expression of ...

African Journals Online (AJOL)

Purpose: To investigate the enhancement of streptokinase extracellular expression in Escherichia coli by adjusting culture media. Methods: Screening of 10 chemical factors (EDTA, peptone, glycine, triton X-100, glycerol, K2HPO4,. KH2PO4, Ca2+ (calcium chloride), yeast and NaCl) in order to increase the secretion of ...

Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

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Gerd Krause

Full Text Available The hormone thyrotropin (TSH and its receptor (TSHR are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR and lutropin/choriogonadotropin (LHR and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One major reason for these open questions is the lack of any structural information about the extracellular segment of TSHR that connects the N-terminal leucine-rich repeat domain (LRRD with the transmembrane helix (TMH 1, the hinge region. It has been shown experimentally that this segment is important for fine tuning of signaling and ligand interactions. A new crystal structure containing most of the extracellular hFSHR region in complex with hFSH has recently been published. Now, we have applied these new structural insights to the homologous TSHR and have generated a structural model of the TSHR LRRD/hinge-region/TSH complex. This structural model is combined and evaluated with experimental data including hormone binding (bTSH, hTSH, thyrostimulin, super-agonistic effects, antibody interactions and signaling regulation. These studies and consideration of significant and non-significant amino acids have led to a new description of mechanisms at the TSHR, including ligand-induced displacements of specific hinge region fragments. This event triggers conformational changes at a convergent center of the LRRD and the hinge region, activating an "intramolecular agonistic unit" close to the transmembrane domain.

Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

Science.gov (United States)

Krause, Gerd; Kreuchwig, Annika; Kleinau, Gunnar

2012-01-01

The hormone thyrotropin (TSH) and its receptor (TSHR) are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR) and lutropin/choriogonadotropin (LHR) and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One major reason for these open questions is the lack of any structural information about the extracellular segment of TSHR that connects the N-terminal leucine-rich repeat domain (LRRD) with the transmembrane helix (TMH) 1, the hinge region. It has been shown experimentally that this segment is important for fine tuning of signaling and ligand interactions. A new crystal structure containing most of the extracellular hFSHR region in complex with hFSH has recently been published. Now, we have applied these new structural insights to the homologous TSHR and have generated a structural model of the TSHR LRRD/hinge-region/TSH complex. This structural model is combined and evaluated with experimental data including hormone binding (bTSH, hTSH, thyrostimulin), super-agonistic effects, antibody interactions and signaling regulation. These studies and consideration of significant and non-significant amino acids have led to a new description of mechanisms at the TSHR, including ligand-induced displacements of specific hinge region fragments. This event triggers conformational changes at a convergent center of the LRRD and the hinge region, activating an "intramolecular agonistic unit" close to the transmembrane domain.

[Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

Science.gov (United States)

Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

2013-01-01

After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

The Calcium-Sensing Receptor in Health and Disease.

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Díaz-Soto, G; Rocher, A; García-Rodríguez, C; Núñez, L; Villalobos, C

2016-01-01

The extracellular calcium-sensing receptor (CaSR) is a unique G protein-coupled receptor (GPCR) activated by extracellular Ca 2+ and by other physiological cations including Mg 2+ , amino acids, and polyamines. CaSR is the most important master controller of the extracellular Ca 2+ homeostatic system being expressed at high levels in the parathyroid gland, kidney, gut and bone, where it regulates parathyroid hormone (PTH) secretion, vitamin D synthesis, and Ca 2+ absorption and resorption, respectively. Gain and loss of function mutations in the CaSR are responsible for severe disturbances in extracellular Ca 2+ metabolism. CaSR agonists (calcimimetics) and antagonists (calcilytics) are in use or under intense research for treatment of hyperparathyroidism secondary to kidney failure and hypocalcemia with hypercalciuria, respectively. Expression of the CaSR extends to other tissues and systems beyond the extracellular Ca 2+ homeostatic system including the cardiovascular system, the airways, and the nervous system where it may play physiological functions yet to be fully understood. As a consequence, CaSR has been recently involved in different pathologies including uncontrolled blood pressure, vascular calcification, asthma, and Alzheimer's disease. Finally, the CaSR has been shown to play a critical role in cancer either contributing to bone metastasis and/or acting as a tumor suppressor in some forms of cancer (parathyroid cancer, colon cancer, and neuroblastoma) and as oncogene in others (breast and prostate cancers). Here we review the role of CaSR in health and disease in calciotropic tissues and others beyond the extracellular calcium homeostatic system. Copyright © 2016 Elsevier Inc. All rights reserved.

Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

Energy Technology Data Exchange (ETDEWEB)

Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

2010-09-21

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

The biphasic effect of extracellular glucose concentration on carbachol-induced fluid secretion from mouse submandibular glands.

Science.gov (United States)

Terachi, Momomi; Hirono, Chikara; Kitagawa, Michinori; Sugita, Makoto

2018-06-01

Cholinergic agonists evoke elevations of the cytoplasmic free-calcium concentration ([Ca 2+ ] i ) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an ex vivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5 mM and elevating it to higher than 5 mM resulted in decreased CCh-induced fluid secretion. The CCh-induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium-glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1-mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl - secretion in real-time. High extracellular glucose levels may suppress the CCh-induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca 2+ ] i signals. © 2018 Eur J Oral Sci.

Effects of low-dose ionising radiation on pituitary adenoma: is there a role for L-type calcium channel?

Energy Technology Data Exchange (ETDEWEB)

Soares, Marcella Araugio; Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia]. E-mail: santosr@cdtn.br

2005-10-15

Pituitary adenomas constitute about 6-18% of brain tumours in adults. Activation of voltage gated calcium currents can account for growth hormone over secretion in some GH-secreting pituitary adenomas that produce an acromegaly appearance and increase mortality. Ca{sup 2+} ions, as mediators of intracellular signalling, are crucial for the development of apoptosis. However, the role of [Ca{sup 2+}] in the development of apoptosis is ambiguous. In this study, the effects of low-dose ionising gamma radiation ({sup 60} Co) on rat pituitary adenoma cells survival and proliferation and the role of calcium channels on the apoptosis radio-induced were evaluated. Doses as low as 3 Gy were found to inhibit GH3 cell proliferation. Even though there was a significant number of live cells,168 hours following irradiation, they were not able to proliferate. The results indicate that the blockade of extracellular calcium influx through these channels does not interfere in the radiation-induced apoptosis in GH3 cells. (author)

Calcium in plant cells

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V. V. Schwartau

2014-04-01

Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

Calcium signaling during reproduction and biotrophic fungal interactions in plants.

Science.gov (United States)

Chen, Junyi; Gutjahr, Caroline; Bleckmann, Andrea; Dresselhaus, Thomas

2015-04-01

Many recent studies have indicated that cellular communications during plant reproduction, fungal invasion, and defense involve identical or similar molecular players and mechanisms. Indeed, pollen tube invasion and sperm release shares many common features with infection of plant tissue by fungi and oomycetes, as a tip-growing intruder needs to communicate with the receptive cells to gain access into a cell and tissue. Depending on the compatibility between cells, interactions may result in defense, invasion, growth support, or cell death. Plant cells stimulated by both pollen tubes and fungal hyphae secrete, for example, small cysteine-rich proteins and receptor-like kinases are activated leading to intracellular signaling events such as the production of reactive oxygen species (ROS) and the generation of calcium (Ca(2+)) transients. The ubiquitous and versatile second messenger Ca(2+) thereafter plays a central and crucial role in modulating numerous downstream signaling processes. In stimulated cells, it elicits both fast and slow cellular responses depending on the shape, frequency, amplitude, and duration of the Ca(2+) transients. The various Ca(2+) signatures are transduced into cellular information via a battery of Ca(2+)-binding proteins. In this review, we focus on Ca(2+) signaling and discuss its occurrence during plant reproduction and interactions of plant cells with biotrophic filamentous microbes. The participation of Ca(2+) in ROS signaling pathways is also discussed. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

The calcium-sensing receptor changes cell shape via a beta-arrestin-1 ARNO ARF6 ELMO protein network.

Science.gov (United States)

Bouschet, Tristan; Martin, Stéphane; Kanamarlapudi, Venkateswarlu; Mundell, Stuart; Henley, Jeremy M

2007-08-01

G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves beta-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative beta-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, beta-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter beta-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, beta-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of beta-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation.

Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales.

Directory of Open Access Journals (Sweden)

Lucía eLopez

2012-09-01

Full Text Available Many natural phenomena display "self-organized criticality'' (SOC. This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium Ca2+ signals, a key component of the signaling toolkit of almost any cell type. Ca2+ signals can either be spatially restricted (local or propagate throughout the cell (global. Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of self-organized criticality. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals ("puffs'' observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not.

Extracellular secretion of recombinant proteins

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Linger, Jeffrey G.; Darzins, Aldis

2014-07-22

Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

PI3K/Akt1 signalling specifies foregut precursors by generating regionalized extra-cellular matrix

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Villegas, S Nahuel; Rothová, Michaela; Barrios-Llerena, Martin E

2013-01-01

-to-mesenchymal transition (EMT). Akt1 transduced this activity via modifications to the extracellular matrix (ECM) and appropriate ECM could itself induce anterior endodermal identity in the absence of PI3K signalling. PI3K/Akt1-modified ECM contained low levels of Fibronectin (Fn1) and we found that Fn1 dose was key...... to specifying anterior endodermal identity in vivo and in vitro. Thus, localized PI3K activity affects ECM composition and ECM in turn patterns the endoderm. DOI: http://dx.doi.org/10.7554/eLife.00806.001....

Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength

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Hansen, Emilie Louise; Sozer, Esin Bengisu; Romeo, Stefania

2015-01-01

death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. METHODS: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability...... was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. RESULTS: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p...-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field. GENERAL SIGNIFICANCE: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials....

Characteristics of calcium signaling in astrocytes induced by photostimulation with femtosecond laser

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Zhao, Yuan; Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zeng, Shaoqun; Luo, Qingming

2010-05-01

Astrocytes have been identified to actively contribute to brain functions through Ca2+ signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca2+) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca2+ signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca2+ wave throughout the network with reduced speed is induced, and intracellular Ca2+ oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca2+ elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca2+ signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca2+ signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.

Calcium as a cardiovascular toxin in CKD-MBD.

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Moe, Sharon M

2017-07-01

Disordered calcium balance and homeostasis are common in patients with chronic kidney disease. Such alterations are commonly associated with abnormal bone remodeling, directly and indirectly. Similarly, positive calcium balance may also be a factor in the pathogenesis of extra skeletal soft tissue and arterial calcification. Calcium may directly affect cardiac structure and function through direct effects to alter cell signaling due to abnormal intracellular calcium homeostasis 2) extra-skeletal deposition of calcium and phosphate in the myocardium and small cardiac arterioles, 3) inducing cardiomyocyte hypertrophy through calcium and hormone activation of NFAT signaling mechanisms, and 4) increased aorta calcification resulting in chronic increased afterload leading to hypertrophy. Similarly, calcium may alter vascular smooth muscle cell function and affect cell signaling which may predispose to a proliferative phenotype important in arteriosclerosis and arterial calcification. Thus, disorders of calcium balance and homeostasis due to CKD-MBD may play a role in the high cardiovascular burden observed in patients with CKD. Published by Elsevier Inc.

Voltage-gated calcium flux mediates Escherichia coli mechanosensation.

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Bruni, Giancarlo N; Weekley, R Andrew; Dodd, Benjamin J T; Kralj, Joel M

2017-08-29

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli , including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

An algorithm for modularization of MAPK and calcium signaling pathways: comparative analysis among different species.

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Nayak, Losiana; De, Rajat K

2007-12-01

Signaling pathways are large complex biochemical networks. It is difficult to analyze the underlying mechanism of such networks as a whole. In the present article, we have proposed an algorithm for modularization of signal transduction pathways. Unlike studying a signaling pathway as a whole, this enables one to study the individual modules (less complex smaller units) easily and hence to study the entire pathway better. A comparative study of modules belonging to different species (for the same signaling pathway) has been made, which gives an overall idea about development of the signaling pathways over the taken set of species of calcium and MAPK signaling pathways. The superior performance, in terms of biological significance, of the proposed algorithm over an existing community finding algorithm of Newman [Newman MEJ. Modularity and community structure in networks. Proc Natl Acad Sci USA 2006;103(23):8577-82] has been demonstrated using the aforesaid pathways of H. sapiens.

Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Modulates Purinoceptor-Coupled Calcium Signaling and Murine Nociceptive Behavior.

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Liu, Pei-Shan; Chueh, Sheau-Huei; Chen, Chin-Chu; Lee, Li-Ya; Shiu, Li-Yen

2017-01-01

Hericium erinaceus is well known for the neurotrophic effect it confers by promoting nerve growth factor biosynthesis. We discovered a novel bioactivity of H. erinaceus in its ability to suppress adenosine triphosphate (ATP)-induced calcium signaling in neuronal PC12 cells. ATP, known primarily as a neurotransmitter, also acts on purinoceptors (P2 purinergic receptor [P2R]) to generate the cellular calcium signaling and secretion that mediate P2R physiological manifestations, including pain. Chronic pain reduces quality of life. However, constant analgesic administration can cause liver and kidney injury, as well as loss of the analgesic effect because of desensitization. In this study we investigated the analgesic potential of H. erinaceus through measurements of ATP-induced Ca2+ signaling in cell lines and observation of pain behaviors in mice. In P2R-coupled Ca2+ signaling measurements, extracts of H. erinaceus mycelia (HEEs) blocked ATP-induced Ca2+ signaling in both rat PC12 cells and human HOS cells. HEEs completely blocked ATP-induced Ca2+ signaling in human HOS cells, suggesting that this effect of HEEs is exerted through the P2R subtypes present in HOS cells, which include the P2X4, P2X7, P2Y2, and P2Y4 subtypes. In observations of animal behavior during pain, HEEs significantly reduced heat-induced pain, including postponing both the tail-flick response to heat stimulation and the paw-lifting response to a hot plate. This study demonstrates novel characteristics of H. erinaceus in reducing nociceptive behavior and blocking the functional activity of P2R. Further studies are required to verify this linkage and its molecular mechanisms.

Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium

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Knudsen, Torben; Johansen, Torben

1989-01-01

1. Pure populations of rat peritoneal mast cells were used to study cellular potassium uptake. The radioactive potassium analogue, 86rubidium, was used as a tracer for potassium for measurements of the activity of the cellular potassium uptake process. 2. The ouabain-sensitive and the ouabain......-resistant potassium (86rubidium) uptake of mast cells incubated in the presence of calcium, 1 mmol l-1, were very low, 52 and 147 pmol per 10(6) cells min-1. 3. Calcium-deprivation of the cells uncovered a large capacity ouabain-sensitive potassium (86rubidium) uptake mechanism. The activity of the uptake mechanism...... was decreased by reintroduction of calcium into the cell suspension, and it was dependent on cellular energy metabolism, temperature and pH. 4. The potassium (86rubidium) uptake of mast cells incubated in a calcium-free medium occurs through an active and ouabain-sensitive mechanism that has the nature...

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

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Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

2016-02-01

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression. Copyright © 2016 by The American Association of Immunologists, Inc.

Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

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Neef, Maren; Ecke, Margret; Hampp, Rüdiger

2015-07-01

In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca^{2+}_{ {cyt}}) concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8 g and μ g during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca^{2+}_{ {cyt}} level during the hypergravity phases of a parabola but a significant increase of the Ca^{2+}_{ {cyt}} concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca^{2+}_{ {cyt}} response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca^{2+}_{ {cyt}} changes entirely. We thus conclude that the significant increase in Ca^{2+}_{ {cyt}} under microgravity is largely due to extracellular Ca2+ sources.

Regulation of cardiomyocyte autophagy by calcium.

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Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

2016-04-15

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

Calcium signaling during the plant-plant interaction of parasitic Cuscuta reflexa with its hosts.

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Albert, Markus; Kaiser, Bettina; van der Krol, Sander; Kaldenhoff, Ralf

2010-09-01

The plant parasite Cuscuta reflexa induces various responses in compatible and incompatible host plants. The visual reactions of both types of host plants including obvious morphological changes require the recognition of Cuscuta ssp. A consequently initiated signaling cascade is triggered which leads to a tolerance of the infection or, in the case of some incompatible host plants, to resistance. Calcium (Ca(2+)) release is the major second messenger during signal transduction. Therefore, we have studied Ca(2+) spiking in tomato and tobacco during infection with C. reflexa. In our recently published study Ca(2+) signals were monitored as bioluminescence in aequorin-expressing tomato plants after the onset of C. reflexa infestation. Signals at the attachment sites were observed from 30 to 48 h after infection. In an assay with leaf disks of aequorin-expressing tomato which were treated with different C. reflexa plant extracts it turned out that the substance that induced Ca(2+) release in the host plant was closely linked to the parasite's haustoria.

Processing and Analysis of Multichannel Extracellular Neuronal Signals: State-of-the-art and Challenges

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Mufti eMahmud

2016-06-01

Full Text Available In recent years multichannel neuronal signal acquisition systems have allowed scientists to focus on research questions which were otherwise impossible. They act as a powerful means to study brain (dysfunctions in in-vivo and in in-vitro animal models. Typically, each session of electrophysiological experiments with multichannel data acquisition systems generate large amount of raw data. For example, a 128 channel signal acquisition system with 16 bits A/D conversion and 20 kHz sampling rate will generate approximately 17 GB data per hour (uncompressed. This poses an important and challenging problem of inferring conclusions from the large amounts of acquired data. Thus, automated signal processing and analysis tools are becoming a key component in neuroscience research, facilitating extraction of relevant information from neuronal recordings in a reasonable time. The purpose of this review is to introduce the reader to the current state-of-the-art of open-source packages for (semiautomated processing and analysis of multichannel extracellular neuronal signals (i.e., neuronal spikes, local field potentials, electroencephalogram, etc., and the existing Neuroinformatics infrastructure for tool and data sharing. The review is concluded by pinpointing some major challenges that are to be faced, which include the development of novel benchmarking techniques, cloud-based distributed processing and analysis tools, as well as defining novel means to share and standardize data.

Fetal brain extracellular matrix boosts neuronal network formation in 3D bioengineered model of cortical brain tissue.

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Sood, Disha; Chwalek, Karolina; Stuntz, Emily; Pouli, Dimitra; Du, Chuang; Tang-Schomer, Min; Georgakoudi, Irene; Black, Lauren D; Kaplan, David L

2016-01-01

The extracellular matrix (ECM) constituting up to 20% of the organ volume is a significant component of the brain due to its instructive role in the compartmentalization of functional microdomains in every brain structure. The composition, quantity and structure of ECM changes dramatically during the development of an organism greatly contributing to the remarkably sophisticated architecture and function of the brain. Since fetal brain is highly plastic, we hypothesize that the fetal brain ECM may contain cues promoting neural growth and differentiation, highly desired in regenerative medicine. Thus, we studied the effect of brain-derived fetal and adult ECM complemented with matricellular proteins on cortical neurons using in vitro 3D bioengineered model of cortical brain tissue. The tested parameters included neuronal network density, cell viability, calcium signaling and electrophysiology. Both, adult and fetal brain ECM as well as matricellular proteins significantly improved neural network formation as compared to single component, collagen I matrix. Additionally, the brain ECM improved cell viability and lowered glutamate release. The fetal brain ECM induced superior neural network formation, calcium signaling and spontaneous spiking activity over adult brain ECM. This study highlights the difference in the neuroinductive properties of fetal and adult brain ECM and suggests that delineating the basis for this divergence may have implications for regenerative medicine.

L-Type Calcium Channels Modulation by Estradiol.

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Vega-Vela, Nelson E; Osorio, Daniel; Avila-Rodriguez, Marco; Gonzalez, Janneth; García-Segura, Luis Miguel; Echeverria, Valentina; Barreto, George E

2017-09-01

Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

Discrete-State Stochastic Models of Calcium-Regulated Calcium Influx and Subspace Dynamics Are Not Well-Approximated by ODEs That Neglect Concentration Fluctuations

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Weinberg, Seth H.; Smith, Gregory D.

2012-01-01

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough (e.g., 10−17 liters) that local signaling may involve 1–100 calcium ions. Is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of fluctuating subspace calcium concentration (a result that accounts for the small subspace volume) with the corresponding deterministic model (an approximation that assumes large system size). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers. PMID:23509597

The microRNA mir-71 inhibits calcium signaling by targeting the TIR-1/Sarm1 adaptor protein to control stochastic L/R neuronal asymmetry in C. elegans.

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Yi-Wen Hsieh

Full Text Available The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON and AWC(OFF, by inhibiting a calcium-mediated signaling pathway in the future AWC(ON cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON, in which mir-71 is expressed at a higher level than in AWC(OFF. In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.

Binding of [125I]iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

International Nuclear Information System (INIS)

Jones, J.I.; Fitzpatrick, L.A.

1990-01-01

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes

NMDAR-mediated calcium transients elicited by glutamate co-release at developing inhibitory synapses

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Abigail Kalmbach

2010-07-01

Full Text Available Before hearing onset, the topographic organization of the inhibitory sound localization pathway from the medial nucleus of the trapezoid body (MNTB to the lateral superior olive (LSO is refined by means of synaptic silencing and strengthening. During this refinement period MNTB-LSO synapses not only release GABA and glycine but also release glutamate. This co-released glutamate can elicit postsynaptic currents that are predominantly mediated by NMDA receptors (NMDARs. To gain a better understanding of how glutamate contributes to synaptic signaling at developing MNTB-LSO inhibitory synapse, we investigated to what degree and under what conditions NMDARs contribute to postsynaptic calcium responses. Our results demonstrate that MNTB-LSO synapses can elicit compartmentalized calcium responses along aspiny LSO dendrites. These responses are significantly attenuated by the NMDARs antagonist APV. APV, however, has no effect on somatically recorded electrical postsynaptic responses, indicating little, if any, contribution of NMDARs to spike generation. Small NMDAR-mediated calcium responses were also observed under physiological levels of extracellular magnesium concentrations indicating that MNTB-LSO synapses activate magnesium sensitive NMDAR on immature LSO dendrites. In Fura-2 AM loaded neurons, blocking GABAA and glycine receptors decreased NMDAR contribution to somatic calcium responses suggesting that GABA and glycine, perhaps by shunting backpropagating action potentials, decrease the level of NMDAR activation under strong stimulus conditions.

Analysis of extracellular RNA by digital PCR

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Kenji eTakahashi

2014-06-01

Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Gap junction modulation by extracellular signaling molecules: the thymus model

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Alves L.A.

2000-01-01

Full Text Available Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.

The Role of Calcium in Osteoporosis

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Arnaud, C. D.; Sanchez, S. D.

1991-01-01

Calcium requirements may vary throughout the lifespan. During the growth years and up to age 25 to 30, it is important to maximize dietary intake of calcium to maintain positive calcium balance and achieve peak bone mass, thereby possibly decreasing the risk of fracture when bone is subsequently lost. Calcium intake need not be greater than 800 mg/day during the relatively short period of time between the end of bone building and the onset of bone loss (30 to 40 years). Starting at age 40 to 50, both men and women lose bone slowly, but women lose bone more rapidly around the menopause and for about 10 years after. Intestinal calcium absorption and the ability to adapt to low calcium diets are impaired in many postmenopausal women and elderly persons owing to a suspected functional or absolute decrease in the ability of the kidney to produce 1,25(OH)2D2. The bones then become more and more a source of calcium to maintain critical extracellular fluid calcium levels. Excessive dietary intake of protein and fiber may induce significant negative calcium balance and thus increase dietary calcium requirements. Generally, the strongest risk factors for osteoporosis are uncontrollable (e.g., sex, age, and race) or less controllable (e.g., disease and medications). However, several factors such as diet, physical activity, cigarette smoking, and alcohol use are lifestyle related and can be modified to help reduce the risk of osteoporosis.

The effect of glucose stimulation on 45calcium uptake of rat pancreatic islets and their total calcium content as measured by a fluorometric micro-method

International Nuclear Information System (INIS)

Wolters, G.H.J.; Wiegman, J.B.; Konijnendijk, W.

1982-01-01

Glucose-stimulated 45 calcium uptake and total calcium content of rat pancreatic islets has been studied, using a new fluorometric micro-method to estimate total calcium. Extracellular calcium was separated from incubated tissue by a rapid micro-filtration procedure. Islets incubated up to 60 min with calcium chloride 2.5 mmol/l and glucose 2.5 mmol/l maintained the same calcium content (670 +- 7.5 pmol/μg DNA). When the glucose concentration was raised to 15 mmol/l no change in the total calcium content could be detected. On incubation with glucose 2.5 mmol/l in the absence of calcium, the calcium content decreased to 488 +- 27 pmol/μg DNA. On incubation with 45 calcium chloride 2.5 mmol/l for 5 or 30 min at 2.5 mmol/l glucose, islets exchanged 21 +- 2 and 28 +- 1% of their total calcium content and, at 15 mmol/l glucose, 30 +- 3 and 45 +- 2%, respectively. Thus, islet calcium has a high turn-over rate. Glucose stimulation results in an increase of the calcium uptake without enhancing the total calcium content and hence must increase the calcium-exchangeable pool. (orig.)

Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.

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Kyle T Beggs

Full Text Available BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor

The danger signal extracellular ATP is an inducer of Fusobacterium nucleatum biofilm dispersal

Directory of Open Access Journals (Sweden)

Qinfeng Ding

2016-11-01

Full Text Available Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP is an example of a danger associated molecular pattern (DAMP employed by mammalian cells to elicit inflammatory and damage healing responses. Although the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were significantly more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also exhibited significantly higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

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Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

2007-01-01

Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

A sensor for calcium uptake

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Collins, Sean; Meyer, Tobias

2011-01-01

Mitochondria — the cell’s power plants — increase their energy production in response to calcium signals in the cytoplasm. A regulator of the elusive mitochondrial calcium channel has now been identified. PMID:20844529

Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

Energy Technology Data Exchange (ETDEWEB)

Kim, Hyunho [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Department of Medicine, University of Maryland, Baltimore, MD (United States); Kang, Ah-Young [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Department of Medicine, Program of Immunology, Graduate School, Seoul National University, Seoul (Korea, Republic of); Ko, Ah-ra [Clinical Research Center, Samsung Biomedical Research Institute, Seoul (Korea, Republic of); Park, Hayne Cho [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); So, Insuk [Department of Physiology, Seoul National University College of Medicine, Seoul (Korea, Republic of); Park, Jong Hoon [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Cheong, Hae Il [Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Pediatrics, Seoul National University Children’s Hospital, Seoul (Korea, Republic of); Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul (Korea, Republic of); Hwang, Young-Hwan [Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Internal Medicine, Eulji General Hospital, Eulji University College of Medicine, Seoul (Korea, Republic of); and others

2014-01-01

Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration. - Highlights: • Polycystin-1 is a target of ubiquitin-independent degradation by calpains. • The PEST domain is required for calpain-mediated degradation of polycystin-1. • Polycystin-1 may independently regulate JAK2 and ERK signaling pathways.

Extracellular vesicles: fundamentals and clinical relevance

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Wael Nassar

2015-01-01

Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

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Purali, Nuhan

2017-09-01

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10 mM NiCl 2 and 10-500 µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

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Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan

2018-05-01

Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

Use of multiple singular value decompositions to analyze complex intracellular calcium ion signals

KAUST Repository

Martinez, Josue G.

2009-12-01

We compare calcium ion signaling (Ca(2+)) between two exposures; the data are present as movies, or, more prosaically, time series of images. This paper describes novel uses of singular value decompositions (SVD) and weighted versions of them (WSVD) to extract the signals from such movies, in a way that is semi-automatic and tuned closely to the actual data and their many complexities. These complexities include the following. First, the images themselves are of no interest: all interest focuses on the behavior of individual cells across time, and thus, the cells need to be segmented in an automated manner. Second, the cells themselves have 100+ pixels, so that they form 100+ curves measured over time, so that data compression is required to extract the features of these curves. Third, some of the pixels in some of the cells are subject to image saturation due to bit depth limits, and this saturation needs to be accounted for if one is to normalize the images in a reasonably un-biased manner. Finally, the Ca(2+) signals have oscillations or waves that vary with time and these signals need to be extracted. Thus, our aim is to show how to use multiple weighted and standard singular value decompositions to detect, extract and clarify the Ca(2+) signals. Our signal extraction methods then lead to simple although finely focused statistical methods to compare Ca(2+) signals across experimental conditions.

Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans

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Singh, Jagmohan; Kumar, Sumit; Phillips, Benjamin

2015-01-01

The extracellular signal that triggers activation of rho-associated kinase (RhoA/ROCK), the major molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone, is not known. Using human IAS tissues, we identified the presence of the biosynthetic machineries for angiotensin II (ANG II), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α). These end products of the renin-angiotensin system (RAS) (ANG II) and arachidonic acid (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were studied. A multipronged approach utilizing immunocytochemistry, Western blot analyses, and force measurements was implemented. Additionally, in a systematic analysis of the effects of respective inhibitors along different steps of biosynthesis and those of antagonists, their end products were evaluated either individually or in combination. To further describe the molecular mechanism for the IAS tone via these pathways, we monitored RhoA/ROCK activation and its signal transduction cascade. Data showed characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS compared with the RSM. Additionally, specific inhibition of the arachidonic acid (AA) pathway caused ∼80% decrease in the IAS tone, whereas that of RAS lead to ∼20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products TXA2, PGF2α, and ANG II, respectively), provide extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS. PMID:25882611

Studies on the production of endogenous pyrogen by rabbit monocytes: the role of calcium and cyclic nucleotides.

OpenAIRE

Sigal, S. L.; Duff, G. W.; Atkins, E.

1985-01-01

Rabbit monocytes stimulated with endotoxin produced endogenous pyrogen, even under conditions of high or low extracellular calcium concentrations. Maximal production occurred when the concentration was in the near-physiological range. Prolonged incubation of cells with a calcium chelator prevented subsequent activation with endotoxin, an effect which was rapidly reversible by re-addition of calcium but not other cations. Addition of small amounts of lanthanum, which acts as a calcium channel ...

Spaceflight Activates Protein Kinase C Alpha Signaling and Modifies the Developmental Stage of Human Neonatal Cardiovascular Progenitor Cells.

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Baio, Jonathan; Martinez, Aida F; Bailey, Leonard; Hasaniya, Nahidh; Pecaut, Michael J; Kearns-Jonker, Mary

2018-02-12

Spaceflight impacts cardiovascular function in astronauts; however, its impact on cardiac development and the stem cells that form the basis for cardiac repair is unknown. Accordingly, further research is needed to uncover the potential relevance of such changes to human health. Using simulated microgravity (SMG) generated by two-dimensional clinorotation and culture aboard the International Space Station (ISS), we assessed the effects of mechanical unloading on human neonatal cardiovascular progenitor cell (CPC) developmental properties and signaling. Following 6-7 days of SMG and 12 days of ISS culture, we analyzed changes in gene expression. Both environments induced the expression of genes that are typically associated with an earlier state of cardiovascular development. To understand the mechanism by which such changes occurred, we assessed the expression of mechanosensitive small RhoGTPases in SMG-cultured CPCs and observed decreased levels of RHOA and CDC42. Given the effect of these molecules on intracellular calcium levels, we evaluated changes in noncanonical Wnt/calcium signaling. After 6-7 days under SMG, CPCs exhibited elevated levels of WNT5A and PRKCA. Similarly, ISS-cultured CPCs exhibited elevated levels of calcium handling and signaling genes, which corresponded to protein kinase C alpha (PKCα), a calcium-dependent protein kinase, activation after 30 days. Akt was activated, whereas phosphorylated extracellular signal-regulated kinase levels were unchanged. To explore the effect of calcium induction in neonatal CPCs, we activated PKCα using hWnt5a treatment on Earth. Subsequently, early cardiovascular developmental marker levels were elevated. Transcripts induced by SMG and hWnt5a-treatment are expressed within the sinoatrial node, which may represent embryonic myocardium maintained in its primitive state. Calcium signaling is sensitive to mechanical unloading and directs CPC developmental properties. Further research both in space and on Earth

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function

International Nuclear Information System (INIS)

Nagayama, Yuji; Wadsworth, H.L.; Chazenbalk, G.D.; Russo, D.; Seto, Pui; Rapoport, B.

1991-01-01

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction the authors constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites

Telocinobufagin inhibits the epithelial-mesenchymal transition of breast cancer cells through the phosphoinositide 3-kinase/protein kinase B/extracellular signal-regulated kinase/Snail signaling pathway.

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Gao, Yuxue; Shi, Lihong; Cao, Zhen; Zhu, Xuetao; Li, Feng; Wang, Ruyan; Xu, Jinyuan; Zhong, Jinyi; Zhang, Baogang; Lu, Shijun

2018-05-01

Telocinobufagin (TBG), an active ingredient of Venenumbufonis , exhibits an immunomodulatory activity. However, its antimetastatic activity in breast cancer remains unknown. The present study investigated whether TBG prevents breast cancer metastasis and evaluated its regulatory mechanism. TBG inhibited the migration and invasion of 4T1 breast cancer cells. Furthermore, TBG triggered the collapse of F-actin filaments in breast cancer. The epithelial-mesenchymal transition (EMT) markers, vimentin and fibronectin, were downregulated following TBG treatment. However, E-cadherin was upregulated following TBG treatment. Snail, a crucial transcriptional factor of EMT, was downregulated following TBG treatment. Signaling pathway markers, including phosphorylated protein kinase B (P-Akt), p-mechanistic target of rapamycin (mTOR) and p-extracellular signal-regulated kinase (ERK), were decreased following TBG treatment. The same results were obtained from in vivo experiments. In conclusion, in vitro and in vivo experiments reveal that TBG inhibited migration, invasion and EMT via the phosphoinositide 3-kinase (PI3K)/Akt/ERK/Snail signaling pathway in breast cancer.

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

Science.gov (United States)

Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

The human Na⁺/H⁺ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

DEFF Research Database (Denmark)

Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

2016-01-01

BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...

Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

Directory of Open Access Journals (Sweden)

Yuko Nakagawa

Full Text Available BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+ ([Ca(2+](c and cAMP ([cAMP](c were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+](c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+](c response. The effect of sucralose on [Ca(2+](c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q inhibitor. Sucralose also induced sustained elevation of [cAMP](c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+ and cAMP-dependent mechanisms.

Extracellular signal-regulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation.

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Pascoli, Vincent; Cahill, Emma; Bellivier, Frank; Caboche, Jocelyne; Vanhoutte, Peter

2014-12-15

Addiction is a chronic and relapsing psychiatric disorder that is thought to occur in vulnerable individuals. Synaptic plasticity evoked by drugs of abuse in the so-called neuronal circuits of reward has been proposed to underlie behavioral adaptations that characterize addiction. By increasing dopamine in the striatum, addictive drugs alter the balance of dopamine and glutamate signals converging onto striatal medium-sized spiny neurons (MSNs) and activate intracellular events involved in long-term behavioral alterations. Our laboratory contributed to the identification of salient molecular changes induced by administration of addictive drugs to rodents. We pioneered the observation that a common feature of addictive drugs is to activate, by a double tyrosine/threonine phosphorylation, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the striatum, which control a plethora of substrates, some of them being critically involved in cocaine-mediated molecular and behavioral adaptations. Herein, we review how the interplay between dopamine and glutamate signaling controls cocaine-induced ERK1/2 activation in MSNs. We emphasize the key role of N-methyl-D-aspartate receptor potentiation by D1 receptor to trigger ERK1/2 activation and its subsequent nuclear translocation where it modulates both epigenetic and genetic processes engaged by cocaine. We discuss how cocaine-induced long-term synaptic and structural plasticity of MSNs, as well as behavioral adaptations, are influenced by ERK1/2-controlled targets. We conclude that a better knowledge of molecular mechanisms underlying ERK1/2 activation by drugs of abuse and/or its role in long-term neuronal plasticity in the striatum may provide a new route for therapeutic treatment in addiction. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Why Calcium? How Calcium Became the Best Communicator*

Science.gov (United States)

Carafoli, Ernesto; Krebs, Joachim

2016-01-01

Calcium carries messages to virtually all important functions of cells. Although it was already active in unicellular organisms, its role became universally important after the transition to multicellular life. In this Minireview, we explore how calcium ended up in this privileged position. Most likely its unique coordination chemistry was a decisive factor as it makes its binding by complex molecules particularly easy even in the presence of large excesses of other cations, e.g. magnesium. Its free concentration within cells can thus be maintained at the very low levels demanded by the signaling function. A large cadre of proteins has evolved to bind or transport calcium. They all contribute to buffer it within cells, but a number of them also decode its message for the benefit of the target. The most important of these “calcium sensors” are the EF-hand proteins. Calcium is an ambivalent messenger. Although essential to the correct functioning of cell processes, if not carefully controlled spatially and temporally within cells, it generates variously severe cell dysfunctions, and even cell death. PMID:27462077

Release of ATP from Marginal Cells in the Cochlea of Neonatal Rats Can Be Induced by Changes in Extracellular and Intracellular Ion Concentrations

Science.gov (United States)

Peng, Yating; Chen, Jie; He, Shan; Yang, Jun; Wu, Hao

2012-01-01

Background Adenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. Methods Sprague-Dawley rats aged 1–3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. Results Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K+ and intra- and extracellular Ca2+. Furthermore, changes in the concentration of intracellular Ca2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. Conclusion We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A2. PMID:23071731

Link between the early calcium deposition in placenta and ...

Indian Academy of Sciences (India)

Administrator

Physiological calcification occurs in bone when the soft extra-cellular matrix is converted into a rigid material capable of sustaining mechanical force. Pathological calcification may occur in different soft tissues that are not associated with calcium deposition for their physiological functions. Moreover, this pathological ...

Changes in force and calcium sensitivity in the developing avian heart.

Science.gov (United States)

Godt, R E; Fogaça, R T; Nosek, T M

1991-11-01

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)

Multistability in a neuron model with extracellular potassium dynamics

Science.gov (United States)

Wu, Xing-Xing; Shuai, J. W.

2012-06-01

Experiments show a primary role of extracellular potassium concentrations in neuronal hyperexcitability and in the generation of epileptiform bursting and depolarization blocks without synaptic mechanisms. We adopt a physiologically relevant hippocampal CA1 neuron model in a zero-calcium condition to better understand the function of extracellular potassium in neuronal seizurelike activities. The model neuron is surrounded by interstitial space in which potassium ions are able to accumulate. Potassium currents, Na+-K+ pumps, glial buffering, and ion diffusion are regulatory mechanisms of extracellular potassium. We also consider a reduced model with a fixed potassium concentration. The bifurcation structure and spiking frequency of the two models are studied. We show that, besides hyperexcitability and bursting pattern modulation, the potassium dynamics can induce not only bistability but also tristability of different firing patterns. Our results reveal the emergence of the complex behavior of multistability due to the dynamical [K+]o modulation on neuronal activities.

Calcium regulates caveolin-1 expression at the transcriptional level

International Nuclear Information System (INIS)

Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming; Li, Yan; Liu, Dan; Zhang, Xue-Cheng; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

2012-01-01

Highlights: ► Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. ► An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. ► Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. ► Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca 2+ /calcineurin/NFAT.

Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy

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Michael eMutlak

2015-07-01

Full Text Available Cardiac hypertrophy results from increased mechanical load on the heart and through the actions of local and systemic neuro-humoral factors, cytokines and growth factors. These mechanical and neuroendocrine effectors act through stretch, G protein-coupled receptors and tyrosine kinases to induce the activation of a myriad of intracellular signaling pathways including the extracellular signal-regulated kinases 1/2 (ERK1/2. Since most stimuli that provoke myocardial hypertrophy also elicit an acute phosphorylation of the threonine-glutamate-tyrosine (TEY motif within the activation loops of ERK1 and ERK2 kinases, resulting in their activation, ERKs have long been considered promotors of cardiac hypertrophy. Several mouse models were generated in order to directly understand the causal role of ERK1/2 activation in the heart. These models include direct manipulation of ERK1/2 such as overexpression, mutagenesis or knockout models, manipulations of upstream kinases such as MEK1 and manipulations of the phosphatases that depohosphorylate ERK1/2 such as DUSP6. The emerging understanding from these studies, as will be discussed here, is more complex than originally considered. While there is little doubt that ERK1/2 activation or the lack of it modulates the hypertrophic process or the type of hypertrophy that develops, it appears that not all ERK1/2 activation events are the same. While much has been learned, some questions remain regarding the exact role of ERK1/2 in the heart, the upstream events that result in ERK1/2 activation and the downstream effector in hypertrophy.

Unilateral vestibular deafferentation-induced changes in calcium signaling-related molecules in the rat vestibular nuclear complex.

Science.gov (United States)

Masumura, Chisako; Horii, Arata; Mitani, Kenji; Kitahara, Tadashi; Uno, Atsuhiko; Kubo, Takeshi

2007-03-23

Inquiries into the neurochemical mechanisms of vestibular compensation, a model of lesion-induced neuronal plasticity, reveal the involvement of both voltage-gated Ca(2+) channels (VGCC) and intracellular Ca(2+) signaling. Indeed, our previous microarray analysis showed an up-regulation of some calcium signaling-related genes such as the alpha2 subunit of L-type calcium channels, calcineurin, and plasma membrane Ca(2+) ATPase 1 (PMCA1) in the ipsilateral vestibular nuclear complex (VNC) following unilateral vestibular deafferentation (UVD). To further elucidate the role of calcium signaling-related molecules in vestibular compensation, we used a quantitative real-time polymerase chain reaction (PCR) method to confirm the microarray results and investigated changes in expression of these molecules at various stages of compensation (6 h to 2 weeks after UVD). We also investigated the changes in gene expression during Bechterew's phenomenon and the effects of a calcineurin inhibitor on vestibular compensation. Real-time PCR showed that genes for the alpha2 subunit of VGCC, PMCA2, and calcineurin were transiently up-regulated 6 h after UVD in ipsilateral VNC. A subsequent UVD, which induced Bechterew's phenomenon, reproduced a complete mirror image of the changes in gene expressions of PMCA2 and calcineurin seen in the initial UVD, while the alpha2 subunit of VGCC gene had a trend to increase in VNC ipsilateral to the second lesion. Pre-treatment by FK506, a calcineurin inhibitor, decelerated the vestibular compensation in a dose-dependent manner. Although it is still uncertain whether these changes in gene expression are causally related to the molecular mechanisms of vestibular compensation, this observation suggests that after increasing the Ca(2+) influx into the ipsilateral VNC neurons via up-regulated VGCC, calcineurin may be involved in their synaptic plasticity. Conversely, an up-regulation of PMCA2, a brain-specific Ca(2+) pump, would increase an efflux of Ca

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine

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Sabine Buhner

2017-11-01

Full Text Available Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations.Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%.Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39 peaking 90 s (64/144 after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9. Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15 in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1–3.Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare.

Redox signaling in acute pancreatitis

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Pérez, Salvador; Pereda, Javier; Sabater, Luis; Sastre, Juan

2015-01-01

Acute pancreatitis is an inflammatory process of the pancreatic gland that eventually may lead to a severe systemic inflammatory response. A key event in pancreatic damage is the intracellular activation of NF-κB and zymogens, involving also calcium, cathepsins, pH disorders, autophagy, and cell death, particularly necrosis. This review focuses on the new role of redox signaling in acute pancreatitis. Oxidative stress and redox status are involved in the onset of acute pancreatitis and also in the development of the systemic inflammatory response, being glutathione depletion, xanthine oxidase activation, and thiol oxidation in proteins critical features of the disease in the pancreas. On the other hand, the release of extracellular hemoglobin into the circulation from the ascitic fluid in severe necrotizing pancreatitis enhances lipid peroxidation in plasma and the inflammatory infiltrate into the lung and up-regulates the HIF–VEGF pathway, contributing to the systemic inflammatory response. Therefore, redox signaling and oxidative stress contribute to the local and systemic inflammatory response during acute pancreatitis. PMID:25778551

Redox signaling in acute pancreatitis

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Salvador Pérez

2015-08-01

Full Text Available Acute pancreatitis is an inflammatory process of the pancreatic gland that eventually may lead to a severe systemic inflammatory response. A key event in pancreatic damage is the intracellular activation of NF-κB and zymogens, involving also calcium, cathepsins, pH disorders, autophagy, and cell death, particularly necrosis. This review focuses on the new role of redox signaling in acute pancreatitis. Oxidative stress and redox status are involved in the onset of acute pancreatitis and also in the development of the systemic inflammatory response, being glutathione depletion, xanthine oxidase activation, and thiol oxidation in proteins critical features of the disease in the pancreas. On the other hand, the release of extracellular hemoglobin into the circulation from the ascitic fluid in severe necrotizing pancreatitis enhances lipid peroxidation in plasma and the inflammatory infiltrate into the lung and up-regulates the HIF–VEGF pathway, contributing to the systemic inflammatory response. Therefore, redox signaling and oxidative stress contribute to the local and systemic inflammatory response during acute pancreatitis.

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

DEFF Research Database (Denmark)

Jorgensen, N R; Geist, S T; Civitelli, R

1997-01-01

mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein......Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium...... stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...

Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling.

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Dan Lv

Full Text Available MHC class I (MHC-I molecules are important components of the immune system. Recently MHC-I have been reported to also play important roles in brain development and synaptic plasticity. In this study, we examine the molecular mechanism(s underlying activity-dependent MHC-I expression using hippocampal neurons. Here we report that neuronal expression level of MHC-I is dynamically regulated during hippocampal development after birth in vivo. Kainic acid (KA treatment significantly increases the expression of MHC-I in cultured hippocampal neurons in vitro, suggesting that MHC-I expression is regulated by neuronal activity. In addition, KA stimulation decreased the expression of pre- and post-synaptic proteins. This down-regulation is prevented by addition of an MHC-I antibody to KA treated neurons. Further studies demonstrate that calcium-dependent protein kinase C (PKC is important in relaying KA simulation activation signals to up-regulated MHC-I expression. This signaling cascade relies on activation of the MAPK pathway, which leads to increased phosphorylation of CREB and NF-κB p65 while also enhancing the expression of IRF-1. Together, these results suggest that expression of MHC-I in hippocampal neurons is driven by Ca2+ regulated activation of the MAPK signaling transduction cascade.

Calcium-Induced Activation of a Mutant G-Protein-Coupled Receptor Causes In Vitro Transformation of NIH/3T3 Cells

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Ana O. Hoff

1999-12-01

Full Text Available The calcium-sensing receptor (CaR is a G-proteincoupled receptor that is widely expressed, has tissuespecific functions, regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151 Met in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

Syndecans as receptors and organizers of the extracellular matrix

DEFF Research Database (Denmark)

Xian, Xiaojie; Gopal, Sandeep; Couchman, John

2009-01-01

, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins...... and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles...

ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

Science.gov (United States)

Zhang, Xinxin; Tang, Xuexi; Wang, Ming; Zhang, Wei; Zhou, Bin; Wang, You

2017-08-01

UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m -2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO 4 3- uptake was more serious compared to NO 3 - uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca 2+ -ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca 2+ -ATPase suppression, and a relation between Ca 2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the

Paracrine signaling in a bacterium.

Science.gov (United States)

López, Daniel; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2009-07-15

Cellular differentiation is triggered by extracellular signals that cause target cells to adopt a particular fate. Differentiation in bacteria typically involves autocrine signaling in which all cells in the population produce and respond to the same signal. Here we present evidence for paracrine signaling in bacterial populations-some cells produce a signal to which only certain target cells respond. Biofilm formation in Bacillus involves two centrally important signaling molecules, ComX and surfactin. ComX triggers the production of surfactin. In turn, surfactin causes a subpopulation of cells to produce an extracellular matrix. Cells that produced surfactin were themselves unable to respond to it. Likewise, once surfactin-responsive cells commenced matrix production, they no longer responded to ComX and could not become surfactin producers. Insensitivity to ComX was the consequence of the extracellular matrix as mutant cells unable to make matrix responded to both ComX and surfactin. Our results demonstrate that extracellular signaling was unidirectional, with one subpopulation producing a signal and a different subpopulation responding to it. Paracrine signaling in a bacterial population ensures the maintenance, over generations, of particular cell types even in the presence of molecules that would otherwise cause those cells to differentiate into other cell types.

Calcium channel blockers and Alzheimer's disease★

Science.gov (United States)

Tan, Yi; Deng, Yulin; Qing, Hong

2012-01-01

Alzheimer's disease is characterized by two pathological hallmarks: amyloid plaques and neurofibrillary tangles. In addition, calcium homeostasis is disrupted in the course of human aging. Recent research shows that dense plaques can cause functional alteration of calcium signals in mice with Alzheimer's disease. Calcium channel blockers are effective therapeutics for treating Alzheimer's disease. This review provides an overview of the current research of calcium channel blockers involved in Alzheimer's disease therapy. PMID:25767489

ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling.

Science.gov (United States)

Weckwerth, Philipp; Ehlert, Britta; Romeis, Tina

2015-03-01

Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize. © 2014 John Wiley & Sons Ltd.

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

Energy Technology Data Exchange (ETDEWEB)

Chen, Qiang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); The First Affiliated Hospital of Xiamen University, Xiamen (China); Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China); Yu, Chundong, E-mail: cdyu@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen (China)

2011-06-17

Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

International Nuclear Information System (INIS)

Chen, Qiang; Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang; Yu, Chundong

2011-01-01

Highlights: → Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. → FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. → FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. → FGFR4-ECD reduced tetracycline-induced fatty liver in mice. → FGFR4-ECD partially restored tetracycline-repressed PPARα expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor α (PPARα), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

Calcium transport across the membrane of Paramecium caudatum (protozoa)

International Nuclear Information System (INIS)

Martinac, B.

1980-06-01

Calcium transport across the membrane of Paramecium caudatum was studied by measuring calcium uptake and release by means of flow-through-technique, which was developed especially for this purpose. The method allows continuous flow of the cells suspension with radioactive and inactive solution, respectively, combined with simultaneous electrical stimulation of the cells by means of extracellular electrodes. The results obtained were compared to and interpreted according to behavioral patterns of Paramecium, which were registered by the time exposure dark-field macrophotographic technique under the same experimental conditions. (orig.) [de

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

Energy Technology Data Exchange (ETDEWEB)

Hansen, R K; Bissell, M J

2000-06-01

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.

Dance band on the Titanic: biomechanical signaling in cardiac hypertrophy.

Science.gov (United States)

Sussman, Mark A; McCulloch, Andrew; Borg, Thomas K

2002-11-15

Biomechanical signaling is a complex interaction of both intracellular and extracellular components. Both passive and active components are involved in the extracellular environment to signal through specific receptors to multiple signaling pathways. This review provides an overview of extracellular matrix, specific receptors, and signaling pathways for biomechanical stimulation in cardiac hypertrophy.

Why Calcium? How Calcium Became the Best Communicator.

Science.gov (United States)

Carafoli, Ernesto; Krebs, Joachim

2016-09-30

Calcium carries messages to virtually all important functions of cells. Although it was already active in unicellular organisms, its role became universally important after the transition to multicellular life. In this Minireview, we explore how calcium ended up in this privileged position. Most likely its unique coordination chemistry was a decisive factor as it makes its binding by complex molecules particularly easy even in the presence of large excesses of other cations, e.g. magnesium. Its free concentration within cells can thus be maintained at the very low levels demanded by the signaling function. A large cadre of proteins has evolved to bind or transport calcium. They all contribute to buffer it within cells, but a number of them also decode its message for the benefit of the target. The most important of these "calcium sensors" are the EF-hand proteins. Calcium is an ambivalent messenger. Although essential to the correct functioning of cell processes, if not carefully controlled spatially and temporally within cells, it generates variously severe cell dysfunctions, and even cell death. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of ethionine on hepatic mitochondrial and microsomal calcium uptake

International Nuclear Information System (INIS)

Agarwal, A.K.; Zinermon, W.D.; Latoni, L.

1988-01-01

Ethionine, an ethyl analog of methionine, produces a variety of physiological and pathological effects in animals. These range from acute effects in the liver, kidney, pancreas, and other organs to liver carcinogenesis. Female rats when injected with ethionine exhibit a rapid decrease in hepatic adenosine triphosphate levels followed by a marked inhibition of RNA and protein synthesis and accumulation of triglycerides. Since calcium transport in mitochondria and microsomes is ATP dependent, it becomes interesting to find out if ethionine administration has any effect on subcellular calcium transport. Calcium has recently gained an increased controversy regarding its role in chemical induced lethal cell damage. Certain groups believe that influx of extracellular calcium across the damaged plasma membrane might actually mediate the irreversible damage to the cell, whereas according to other, entry of calcium into the cell is secondary to the damage. The present study was carried out to investigate the calcium [ 45 Ca] transport in mitochondria and microsomes following ethionine administration. The effect of carbon tetrachloride on calcium uptake in ethionine treated rats was also studied

Calcium dynamics in vascular smooth muscle

OpenAIRE

Amberg, Gregory C.; Navedo, Manuel F.

2013-01-01

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells....

Integrins and extracellular matrix in mechanotransduction

Directory of Open Access Journals (Sweden)

Ramage L

2011-12-01

Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

Arsenic trioxide (As2O3) induced calcium signals and cytotoxicity in two human cell lines: SY-5Y neuroblastoma and 293 embryonic kidney (HEK)

International Nuclear Information System (INIS)

Florea, Ana-Maria; Splettstoesser, Frank; Buesselberg, Dietrich

2007-01-01

Arsenic trioxide (As 2 O 3 ) has anticancer properties; however, its use also leads to neuro-, hepato- or nephro-toxicity, and therefore, it is important to understand the mechanism of As 2 O 3 toxicity. We studied As 2 O 3 influence on intracellular calcium ([Ca 2+ ] i ) homeostasis of human neuroblastoma SY-5Y and embryonic kidney cells (HEK 293).We also relate the As 2 O 3 induced [Ca 2+ ] i modifications with cytotoxicity. We used Ca 2+ sensitive dyes (fluo-4 and rhod-2) combined with laser scanning microscopy or fluorescence activated cell sorting to measure Ca 2+ changes during the application of As 2 O 3 and we approach evaluation of cytotoxicity. As 2 O 3 (1 μM) increased [Ca 2+ ] i in SY-5Y and HEK 293 cells. Three forms of [Ca 2+ ] i -elevations were found: (1) steady-state increases (2) transient [Ca 2+ ] i -elevations and (3) Ca 2+ -spikes. [Ca 2+ ] i modifications were independent from extracellular Ca 2+ but dependent on internal calcium stores. The effect was not reversible. Inositol triphosphate (IP 3 ) and ryanodine (Ry) receptors are involved in regulation of signals induced by As 2 O 3 . 2-APB and dantrolene significantly reduced the [Ca 2+ ] i -rise (p 2+ ] i -elevation or spiking. This indicates that other Ca 2+ regulating mechanisms are involved. In cytotoxicity tests As 2 O 3 significantly reduced cell viability in both cell types. Staining with Hoechst 33342 showed occurrence of apoptosis and DNA damage. Our data suggest that [Ca 2+ ] i is an important messenger in As 2 O 3 induced cell death

Characterization of NAADP-mediated calcium signaling in human spermatozoa

Energy Technology Data Exchange (ETDEWEB)

Sánchez-Tusie, A.A. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Vasudevan, S.R.; Churchill, G.C. [Department of Pharmacology, University of Oxford, Oxford OX1 3QT, England (United Kingdom); Nishigaki, T. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Treviño, C.L., E-mail: ctrevino@ibt.unam.mx [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico)

2014-01-10

Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca{sup 2+}]{sub i} increases in human sperm in the absence of [Ca{sup 2+}]{sub o}. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca{sup 2+} signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca{sup 2+}-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca{sup 2+} signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca{sup 2+} and pH. Ca{sup 2+} fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca{sup 2+}] increases in human sperm even in the absence of extracellular Ca{sup 2+}. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

Science.gov (United States)

Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

2015-10-01

Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.

LFPy: A tool for biophysical simulation of extracellular potentials generated by detailed model neurons

Directory of Open Access Journals (Sweden)

Henrik eLindén

2014-01-01

Full Text Available Electrical extracellular recordings, i.e., recordings of the electrical potentials in the extracellular medium between cells, have been a main work-horse in electrophysiology for almost a century. The high-frequency part of the signal (>=500 Hz, i.e., themulti-unit activity (MUA, contains information about the firing of action potentials in surrounding neurons, while the low-frequency part, the local field potential (LFP, contains information about how these neurons integrate synaptic inputs. As the recorded extracellular signals arise from multiple neural processes, their interpretation is typically ambiguous and difficult. Fortunately, a precise biophysical modeling scheme linking activity at the cellular level and the recorded signal has been established: the extracellular potential can be calculated as a weighted sum of all transmembrane currents in all cells located in the vicinity of the electrode. This computational scheme can considerably aid the modeling and analysis of MUA and LFP signals.Here, we describe LFPy, an open source Python package for numerical simulations of extracellular potentials. LFPy consists of a set of easy-to-use classes for defining cells, synapses and recording electrodes as Python objects, implementing this biophysical modeling scheme. It runs on top of the widely used NEURON simulation environment, which allows for flexible usage of both new and existing cell models.Further, calculation of extracellular potentials using the line-source-method is efficiently implemented.We describe the theoretical framework underlying the extracellular potential calculations and illustrate by examples how LFPy can be used both for simulating LFPs, i.e., synaptic contributions from single cells as well a populations of cells, and MUAs, i.e., extracellular signatures of action potentials.

LFPy: a tool for biophysical simulation of extracellular potentials generated by detailed model neurons.

Science.gov (United States)

Lindén, Henrik; Hagen, Espen; Lęski, Szymon; Norheim, Eivind S; Pettersen, Klas H; Einevoll, Gaute T

2013-01-01

Electrical extracellular recordings, i.e., recordings of the electrical potentials in the extracellular medium between cells, have been a main work-horse in electrophysiology for almost a century. The high-frequency part of the signal (≳500 Hz), i.e., the multi-unit activity (MUA), contains information about the firing of action potentials in surrounding neurons, while the low-frequency part, the local field potential (LFP), contains information about how these neurons integrate synaptic inputs. As the recorded extracellular signals arise from multiple neural processes, their interpretation is typically ambiguous and difficult. Fortunately, a precise biophysical modeling scheme linking activity at the cellular level and the recorded signal has been established: the extracellular potential can be calculated as a weighted sum of all transmembrane currents in all cells located in the vicinity of the electrode. This computational scheme can considerably aid the modeling and analysis of MUA and LFP signals. Here, we describe LFPy, an open source Python package for numerical simulations of extracellular potentials. LFPy consists of a set of easy-to-use classes for defining cells, synapses and recording electrodes as Python objects, implementing this biophysical modeling scheme. It runs on top of the widely used NEURON simulation environment, which allows for flexible usage of both new and existing cell models. Further, calculation of extracellular potentials using the line-source-method is efficiently implemented. We describe the theoretical framework underlying the extracellular potential calculations and illustrate by examples how LFPy can be used both for simulating LFPs, i.e., synaptic contributions from single cells as well a populations of cells, and MUAs, i.e., extracellular signatures of action potentials.

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

Science.gov (United States)

Hansen, R K; Bissell, M J

2010-01-01

The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function. PMID:10903527

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Doktycz, Mitchel John [ORNL; Morrell-Falvey, Jennifer L [ORNL

2011-01-01

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

The effect of organolead and -tin compounds on signal transduction in vitro: Investigations on the cytosolic free calcium concentration; Der Einfluss von organischen Blei- und Zinnverbindungen auf die Signaltransduktion in vitro: Untersuchungen zur Veraenderung der zytosolischen freien Calciumkonzentration

Energy Technology Data Exchange (ETDEWEB)

Ade, T.

1996-03-01

The cellular effects of organolead and -tin compounds are not yet precisely understood. However, on the basis of their immuno- and neurotoxicity it is most likely that these substances interfere with cellular signal transduction. For this reason the effect on cytosolic free calcium concentration was investigated in this study. The organometals used induce a persistent increase in cytosolic free calcium concentration in human leukaemia HL-60 cells as well as in neuroblastoma NG-108-15 cells. Studies of the mechanism of the organometal effect with EGTA and calcium channel blockers revealed that an influx of calcium from the extracellular space is responsible for the organometal-induced calcium elevation in HL-60 cells. The effect of the investigated lead compounds and tributyltin is due to calcium channel opening in the plasma membrane. The same is true for the NG108-15 cells. Activation of distinct receptor-mediated signal transduction is not the reason for channel opening. The regulation of cytosolic free calcium concentration was affected by inhibition of plasmamembrane Ca{sup 2+}-ATPases as well as by disturbance of other ion gradients. A consequence of the organometal effect on the cytosolic calcium concentration is the activation of a cPLA{sub 2} and perhaps the induction of apoptosis. These results contribute towards the understanding of biochemical mechanisms causing the injury of vells by organometals. (orig.) [Deutsch] Die zellulaeren Wirkungsmechanismen organischer Blei- und Zinnverbindungen sind zum grossen Teil nicht verstanden. Die immuno- und neurotoxischen Effekte dieser Xenobiotika lassen jedoch die Beeinflussung der Signalwege in den Zellen vermuten. Daher lag der Schwerpunkt dieser Arbeit in der Untersuchung der Signaluebertragungswege und der damit verbundenen Regulation des Calciums. Sowohl in immunkompetenten Zellen (HL-60) wie auch in neuronalen Zellen (NG108-15) induzierten die untersuchten Organometalle eine persistente Erhoehung der

Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane.

Science.gov (United States)

Savino, John A; Evans, Jodi F; Rabinowitz, Dorianne; Auborn, Karen J; Carter, Timothy H

2006-03-01

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We asked whether elevated cytosolic free calcium [Ca2+]i is required for cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2 fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause apoptosis, measured by nucleosome release, under conditions that prevented elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER calcium. C33A and DU145 responded differently to perturbations in Ca2+ homeostasis, suggesting that DIM induces apoptosis by different mechanisms in these two cell lines and/or that calcium mobilization also activates different survival pathways in C33A and DU145. Apoptosis in C33A was independent of increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+ signaling is important for cell survival but the characteristics of this response also differed in the two cell lines. Our results underscore the complex and variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both calcium-dependent and calcium-independent mechanisms.

Traversing the Links between Heavy Metal Stress and Plant Signaling

Science.gov (United States)

Jalmi, Siddhi K.; Bhagat, Prakash K.; Verma, Deepanjali; Noryang, Stanzin; Tayyeba, Sumaira; Singh, Kirti; Sharma, Deepika; Sinha, Alok K.

2018-01-01

Plants confront multifarious environmental stresses widely divided into abiotic and biotic stresses, of which heavy metal stress represents one of the most damaging abiotic stresses. Heavy metals cause toxicity by targeting crucial molecules and vital processes in the plant cell. One of the approaches by which heavy metals act in plants is by over production of reactive oxygen species (ROS) either directly or indirectly. Plants act against such overdose of metal in the environment by boosting the defense responses like metal chelation, sequestration into vacuole, regulation of metal intake by transporters, and intensification of antioxidative mechanisms. This response shown by plants is the result of intricate signaling networks functioning in the cell in order to transmit the extracellular stimuli into an intracellular response. The crucial signaling components involved are calcium signaling, hormone signaling, and mitogen activated protein kinase (MAPK) signaling that are discussed in this review. Apart from signaling components other regulators like microRNAs and transcription factors also have a major contribution in regulating heavy metal stress. This review demonstrates the key role of MAPKs in synchronously controlling the other signaling components and regulators in metal stress. Further, attempts have been made to focus on metal transporters and chelators that are regulated by MAPK signaling. PMID:29459874

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

NARCIS (Netherlands)

Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A; Del Portillo, Hernando A; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Görgens, André; Gupta, Ramesh C; Hendrix, An; Hermann, Dirk M; Hill, Andrew F; Hochberg, Fred; Horn, Peter A; de Kleijn, Dominique; Kordelas, Lambros; Kramer, Boris W; Krämer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J; Lim, Sai Kiang; Lötvall, Jan; Maguire, Casey A; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G; Rivera, Francisco J; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W M; Wauben, Marca|info:eu-repo/dai/nl/112675735; Andaloussi, Samir El; Théry, Clotilde; Rohde, Eva; Giebel, Bernd

2015-01-01

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information

The calcium-sensing receptor and the reproductive system

Directory of Open Access Journals (Sweden)

Isabella Ellinger

2016-08-01

Full Text Available Active placental transport of maternal serum calcium (Ca2+ to the offspring is pivotal for proper development of the fetal skeleton as well as various organ systems. Moreover, extracellular Ca2+ levels impact on distinct processes in mammalian reproduction. The calcium-sensing receptor (CaSR translates changes in extracellular Ca2+-concentrations into cellular reactions. This review summarizes current knowledge on the expression of CaSR and its putative functions in reproductive organs. CaSR was detected in placental cells mediating materno-fetal Ca2+-transport such as the the murine intraplacental yolk sac and the human syncytiotrophoblast. As shown in casr knock-out mice, ablation of CaSR downregulates transplacental Ca2+-transport. Receptor expression was reported in human and rat ovarian surface epithelial cells, where CaSR activation stimulates cell proliferation. In follicles of various species a role of CaSR activation in oocyte maturation was suggested. Based on studies in avian follicles, the activation of CaSR expressed in granulosa cells may support the survival of follicles after their selection. CaSR in rat and equine sperms was functionally linked to sperm motility and sperm capacitation. Implantation involves complex interactions between the blastocyst and the uterine epithelium. During early pregnancy, CaSR expression at the implantation site as well as in decidual cells indicates that CaSR is important for blastocyst implantation and decidualization in the rat uterus. Localization of CaSR in human extravillous cytotrophoblasts suggests a role of CaSR in placentation. Overall, evidence for functional involvement of CaSR in physiologic mammalian reproductive processes exists. Moreover, several studies reported altered expression of CaSR in cells of reproductive tissues under pathologic conditions. However, in many tissues we still lack knowledge on physiological ligands activating CaSR, CaSR-linked G-proteins, activated

Effect of acute acid loading on acid-base and calcium metabolism

DEFF Research Database (Denmark)

Osther, Palle J

2006-01-01

OBJECTIVE: To investigate the acid-base and calcium metabolic responses to acute non-carbonic acid loading in idiopathic calcium stone-formers and healthy males using a quantitative organ physiological approach. MATERIAL AND METHODS: Five-h ammonium chloride loading studies were performed in 12...... male recurrent idiopathic calcium stone-formers and 12 matched healthy men using a randomized, placebo-controlled, cross-over design. Arterialized capillary blood, serum and urine were collected hourly for measurement of electrolytes, ionized calcium, magnesium, phosphate, parathyroid hormone and acid-base...... status. Concentrations of non-metabolizable base (NB) and acid (NA) were calculated from measured concentrations of non-metabolizable ions. RESULTS: The extracellular acid-base status in the stone-formers during basal conditions and acid loading was comparable to the levels in the healthy controls...

Purinergic Signalling in Inflammatory Renal Disease

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Nishkantha eArulkumaran

2013-07-01

Full Text Available Extracellular purines have a role in renal physiology and adaption to inflammation. However, inflammatory renal disease may be mediated by extracellular purines, resulting in renal injury. The role of purinergic signalling is dependent on the concentrations of extracellular purines. Low basal levels of purines are important in normal homeostasis and growth. Concentrations of extracellular purines are significantly elevated during inflammation and mediate either an adaptive role or propagate local inflammation. Adenosine signalling mediates alterations in regional renal blood flow by regulation of the renal microcirculation, tubulo-glomerular feedback, and tubular transport of sodium and water. Increased extracellular ATP and renal P2 receptor-mediated inflammation are associated with various renal diseases, including hypertension, diabetic nephropathy, and glomerulonephritis. Experimental data suggests P2 receptor deficiency or receptor antagonism is associated with amelioration of antibody-mediated nephritis, suggesting a pathogenic (rather than adaptive role of purinergic signalling. We discuss the role of extracellular nucleotides in adaptation to ischaemic renal injury and in the pathogenesis of inflammatory renal disease.

Modeling the intra- and extracellular cytokine signaling pathway under heat stroke in the liver.

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Maria Rodriguez-Fernandez

Full Text Available Heat stroke (HS is a life-threatening illness induced by prolonged exposure to a hot environment that causes central nervous system abnormalities and severe hyperthermia. Current data suggest that the pathophysiological responses to heat stroke may not only be due to the immediate effects of heat exposure per se but also the result of a systemic inflammatory response syndrome (SIRS. The observation that pro- (e.g., IL-1 and anti-inflammatory (e.g., IL-10 cytokines are elevated concomitantly during recovery suggests a complex network of interactions involved in the manifestation of heat-induced SIRS. In this study, we measured a set of circulating cytokine/soluble cytokine receptor proteins and liver cytokine and receptor mRNA accumulation in wild-type and tumor necrosis factor (TNF receptor knockout mice to assess the effect of neutralization of TNF signaling on the SIRS following HS. Using a systems approach, we developed a computational model describing dynamic changes (intra- and extracellular events in the cytokine signaling pathways in response to HS that was fitted to novel genomic (liver mRNA accumulation and proteomic (circulating cytokines and receptors data using global optimization. The model allows integration of relevant biological knowledge and formulation of new hypotheses regarding the molecular mechanisms behind the complex etiology of HS that may serve as future therapeutic targets. Moreover, using our unique modeling framework, we explored cytokine signaling pathways with three in silico experiments (e.g. by simulating different heat insult scenarios and responses in cytokine knockout strains in silico.

Microalgal bacterial flocs treating paper mill effluent: A sunlight-based approach for removing carbon, nitrogen, phosphorus, and calcium.

Science.gov (United States)

Van Den Hende, Sofie; Rodrigues, André; Hamaekers, Helen; Sonnenholzner, Stanislaus; Vervaeren, Han; Boon, Nico

2017-10-25

Treatment of upflow anaerobic sludge blanket (UASB) effluent from a paper mill in aerated activated sludge reactors involves high aeration costs. Moreover, this calcium-rich effluent leads to problematic scale formation. Therefore, a novel strategy for the aerobic treatment of paper mill UASB effluent in microalgal bacterial floc sequencing batch reactors (MaB-floc SBRs) is proposed, in which oxygen is provided via photosynthesis, and calcium is removed via bio-mineralization. Based on the results of batch experiments in the course of this study, a MaB-floc SBR was operated at an initial neutral pH. This SBR removed 58±21% organic carbon, 27±8% inorganic carbon, 77±5% nitrogen, 73±2% phosphorus, and 27±11% calcium. MaB-flocs contained 10±3% calcium, including biologically-influenced calcite crystals. The removal of calcium and inorganic carbon by MaB-flocs significantly decreased when inhibiting extracellular carbonic anhydrase (CA), an enzyme that catalyses the hydration and dehydration of CO 2 . This study demonstrates the potential of MaB-floc SBRs for the alternative treatment of calcium-rich paper mill effluent, and highlights the importance of extracellular CA in this treatment process. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy.

Science.gov (United States)

Edwards, Amanda Nicole; Siuti, Piro; Bible, Amber N; Alexandre, Gladys; Retterer, Scott T; Doktycz, Mitchel J; Morrell-Falvey, Jennifer L

2011-01-01

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition. FEMS Microbiology Letters © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

Pharmacological analysis of calcium transients in response to gravity vector change in Arabidopsis hypocotyls and petioles.

Science.gov (United States)

Toyota, M.; Furuichi, T.; Tatsumi, H.; Sokabe, M.

Plants regulate their growth and morphology in response to gravity field known as gravitropism in general In the process of gravitropism gravity sensing will form the critical earliest event which is supposed to take place in specialized cells statocytes such as columella cells and shoot endodermal cells Although gravistimulation is assumed to be converted into certain intracellular signals the underlying transduction mechanisms have hardly been explored One of the potential candidates for the intracellular signals is an increase in the cytoplasmic free calcium concentration Ca 2 c Here we measured Ca 2 c changes induced by gravistimulation in seedlings of Arabidopsis thaliana expressing aequorin as a calcium reporter When a plate of seedlings was turned through 180 r Ca 2 c transiently increased within 50 s and decayed exponentially with a time constant of ca 60 s The amplitude of the Ca 2 c increase was independent of the angular velocity of the rotation The Ca 2 c increase was reversibly blocked by extracellularly applied potential mechanosensitive channel blockers La 3 Gd 3 or a Ca 2 chelator BAPTA indicating that it arose from Ca 2 -influx via Ca 2 -permeable channel s on the plasma membrane Furthermore the Ca 2 c increase was attenuated by actin-disrupting drugs latrunculin B cytochalasin B but not by microtuble-disrupting drugs oryzalin nocodazole indicating that the activation of

Mechanical signaling coordinates the embryonic heartbeat

Science.gov (United States)

Chiou, Kevin K.; Rocks, Jason W.; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E.; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F.; Prosser, Benjamin L.; Discher, Dennis E.; Liu, Andrea J.

2016-01-01

In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats. PMID:27457951

Signal percolation through plants and the shape of the calcium signature.

Science.gov (United States)

Plieth, Christoph

2010-04-01

Plants respond to almost any kind of external stimulus with transients in their cytoplasmic free calcium concentration ([Ca(2+)](c)). A huge variety of kinetics recorded by optical techniques has been reported in the past. This variety has been credited the specificity needed to explain how information about incoming stimuli is evaluated by the organism and turned into the right physiological responses which provide advantages for survival and reproduction. A physiological response often takes place away from the site of stimulation. This requires cell-to-cell communication. Hence, responding cells are not necessarily directly stimulated but rather receive an indirect stimulus via cell-to-cell communication. It appears unlikely that the '[Ca(2+)](c) signature' in the primarily stimulated cell is conveyed over long distances via cell-to-cell communication from the 'receptor cells' to the 'effector cells'. Here, a novel aspect is highlighted to explain the variety of [Ca(2+)] kinetics seen by integrating methods of [Ca(2+)](c) recording. Plants can generally be seen as cellular automata with specific morphology and capable for cell-to-cell communication. Just a few rules are needed to demonstrate how waves of [Ca(2+)](c)-increases percolate through the organism and thereby deliver a broad variety of 'signatures'. Modelling intercellular signalling may be a possible way to find explanations for different kinds of signal transmission, signal amplification, wave formation, oscillations and stimulus-response coupling. The basic examples presented here show that care has to be taken when interpreting cellular '[Ca(2+)](c) signatures' recorded by optical techniques which integrate over a big number of cells or even whole plants.

Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

Directory of Open Access Journals (Sweden)

Pengchong Jiang

Full Text Available Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme, suramin (broad-spectrum P2 receptor antagonist, 2-APB (IP3 receptor blocker and thapsigargin (endoplasmic reticulum calcium pump inhibitor potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

Calcium currents in a fast-twitch skeletal muscle of the rat.

Science.gov (United States)

Donaldson, P L; Beam, K G

1983-10-01

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

Science.gov (United States)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Effects of calcium, calcium entry blockers and calmodulin inhibitors on atrioventricular conduction disturbances induced by hypoxia.

OpenAIRE

Anno, T.; Kodama, I.; Shibata, S.; Toyama, J.; Yamada, K.

1986-01-01

Effects of hypoxia on atrioventricular conduction were investigated in the Langendorff-perfused isolated heart of the rabbit with various extracellular calcium concentrations ([Ca2+]) as well as in the presence of verapamil, nifedipine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and chlorpromazine. The prolongation of the atrio-His (AH) interval by hypoxia for 7 min was greater with increasing [Ca2+]o ranging from 1.2 to 5.2 mM. At [Ca2+]o of over 3.2 mM under hypoxic condition...

Albumin-induced apoptosis of glomerular parietal epithelial cells is modulated by extracellular signal-regulated kinase 1/2

Science.gov (United States)

Ohse, Takamoto; Krofft, Ron D.; Wu, Jimmy S.; Eddy, Allison A.; Pippin, Jeffrey W.; Shankland, Stuart J.

2012-01-01

Background. The biological role(s) of glomerular parietal epithelial cells (PECs) is not fully understood in health or disease. Given its location, PECs are constantly exposed to low levels of filtered albumin, which is increased in nephrotic states. We tested the hypothesis that PECs internalize albumin and increased uptake results in apoptosis. Methods. Confocal microscopy of immunofluorescent staining and immunohistochemistry were used to demonstrate albumin internalization in PECs and to quantitate albumin uptake in normal mice and rats as well as experimental models of membranous nephropathy, minimal change disease/focal segmental glomerulosclerosis and protein overload nephropathy. Fluorescence-activated cell sorting analysis was performed on immortalized cultured PECs exposed to fluorescein isothiocyanate (FITC)-labeled albumin in the presence of an endosomal inhibitor or vehicle. Apoptosis was measured by Hoechst staining in cultured PECs exposed to bovine serum albumin. Levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) were restored by retroviral infection of mitogen-activated protein kinase (MEK) 1/2 and reduced by U0126 in PECs exposed to high albumin levels in culture and apoptosis measured by Hoechst staining. Results. PECs internalized albumin normally, and this was markedly increased in all of the experimental disease models (P PECs also internalize FITC-labeled albumin, which was reduced by endosomal inhibition. A consequence of increased albumin internalization was PEC apoptosis in vitro and in vivo. Candidate signaling pathways underlying these events were examined. Data showed markedly reduced levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in PECs exposed to high albumin levels in nephropathy and in culture. A role for ERK1/2 in limiting albumin-induced apoptosis was shown by restoring p-ERK1/2 by retroviral infection, which reduced apoptosis in cultured PECs, while a forced

Moringa oleifera-rich diet and T cell calcium signaling in spontaneously hypertensive rats.

Science.gov (United States)

Attakpa, E S; Bertin, G A; Chabi, N W; Ategbo, J-M; Seri, B; Khan, N A

2017-11-24

Moringa oleifera is a plant whose fruits, roots and leaves have been advocated for traditional medicinal uses. The physicochemical analysis shows that Moringa oleifera contains more dietary polyunsaturated fatty acids (PUFA) than saturated fatty acids (SFA). The consumption of an experimental diet enriched with Moringa oleifera extracts lowered blood pressure in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) rats as compared to rats fed an unsupplemented control diet. Anti-CD3-stimulated T cell proliferation was diminished in both strains of rats fed the Moringa oleifera. The experimental diet lowered secretion of interleukin-2 in SHR, but not in WKY rats compared with rats fed the control diet. Studies of platelets from patients with primary hypertension and from SHR support the notion that the concentration of intracellular free calcium [Ca(2+)](i) is modified in both clinical and experimental hypertension. We observed that the basal, [Ca(2+)](i) was lower in T cells of SHR than in those of WKY rats fed the control diet. Feeding the diet with Moringa oleifera extracts to WKY rats did not alter basal [Ca(2+)](i) in T cells but increased basal [Ca(2+)](i) in SHR. Our study clearly demonstrated that Moringa oleifera exerts antihypertensive effects by inhibiting the secretion of IL-2 and modulates T cell calcium signaling in hypertensive rats.

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture*

Science.gov (United States)

Mauceri, Daniela; Hagenston, Anna M.; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-01-01

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. PMID:26231212

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

Energy Technology Data Exchange (ETDEWEB)

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M.; Mroue, Rana; Spencer, Virginia A.; Brownfield, Doug; Radisky, Derek C.; Bustamante, Carlos; Bissell, Mina J.

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.

Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

Directory of Open Access Journals (Sweden)

Margarida Ressurreição

2014-06-01

Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation.

Science.gov (United States)

Stobart, Jillian L; Ferrari, Kim David; Barrett, Matthew J P; Stobart, Michael J; Looser, Zoe J; Saab, Aiman S; Weber, Bruno

2018-01-01

Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2-/-) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The functions of store-operated calcium channels.

Science.gov (United States)

Putney, James W; Steinckwich-Besançon, Natacha; Numaga-Tomita, Takuro; Davis, Felicity M; Desai, Pooja N; D'Agostin, Diane M; Wu, Shilan; Bird, Gary S

2017-06-01

Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca 2+ stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Published by Elsevier B.V.

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

Science.gov (United States)

Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

2005-10-28

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

Science.gov (United States)

Parra, M.; Vercoutere, W.; Roden, C.; Banerjee, I.; Krauser, W.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2003-01-01

We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense

Calcium-sensing receptor: Role in health and disease

Directory of Open Access Journals (Sweden)

R V Thakker

2012-01-01

Full Text Available The calcium-sensing receptor (CaSR is a 1,078 amino acid G protein-coupled receptor (GPCR, which is predominantly expressed in the parathyroids and kidney. The CaSR allows regulation of parathyroid hormone (PTH secretion and renal tubular calcium re-absorption in response to alterations in extracellular calcium concentrations. Loss-of-function CaSR mutations have been reported in the hypercalcemic disorders of familial benign (hypocalciuric hypercalcemia (FBH or FHH, neonatal severe primary hyperparathyroidism (NSHPT, and adult primary hyperparathyroidism. However, some individuals with loss-of-function CaSR mutations remain normocalcemic. Gain-of-function CaSR mutations have been shown to result in autosomal-dominant hypocalcemia with hypercalciuria (ADHH and Bartter′s syndrome type V. CaSR auto-antibodies have been found in FHH patients who did not have loss-of-function CaSR mutations and in patients with an acquired form (i.e. autoimmune of hypoparathyroidism. Thus, abnormalities of the CaSR are associated with 4 hypercalcemic and 3 hypocalcemic disorders.

Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

DEFF Research Database (Denmark)

Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern

2012-01-01

Based on a rare, natural Glu for Ala204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2 (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional....... Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment...

Detection and quantification of coronary calcium from dual energy chest x-rays: Phantom feasibility study.

Science.gov (United States)

Zhou, Bo; Wen, Di; Nye, Katelyn; Gilkeson, Robert C; Eck, Brendan; Jordan, David; Wilson, David L

2017-10-01

We have demonstrated the ability to identify coronary calcium, a reliable biomarker of coronary artery disease, using nongated, 2-shot, dual energy (DE) chest x-ray imaging. Here we will use digital simulations, backed up by measurements, to characterize DE calcium signals and the role of potential confounds such as beam hardening, x-ray scatter, cardiac motion, and pulmonary artery pulsation. For the DE calcium signal, we will consider quantification, as compared to CT calcium score, and visualization. We created stylized and anatomical digital 3D phantoms including heart, lung, coronary calcium, spine, ribs, pulmonary artery, and adipose. We simulated high and low kVp x-ray acquisitions with x-ray spectra, energy dependent attenuation, scatter, ideal detector, and automatic exposure control (AEC). Phantoms allowed us to vary adipose thickness, cardiac motion, etc. We used specialized dual energy coronary calcium (DECC) processing that includes corrections for scatter and beam hardening. Beam hardening over a wide range of adipose thickness (0-30 cm) reduced the change in intensity of a coronary artery calcification (ΔI CAC ) by calcium signal (ΔI CAC ) in DECC images ±9%. If a simulated pulmonary artery fills with blood between exposures, it can give rise to a residual signal in DECC images, explaining pulmonary artery visibility in some clinical images. Residual misregistration can be mostly compensated by integrating signals in an enlarged region encompassing registration artifacts. DECC calcium score compared favorably to CT mass and volume scores over a number of phantom perturbations. Simulations indicate that proper DECC processing can faithfully recover coronary calcium signals. Beam hardening, errors in scatter estimation, cardiac motion, calcium residual misregistration etc., are all manageable. Simulations are valuable as we continue to optimize DE coronary calcium image processing and quantitative analysis. © 2017 American Association of Physicists

Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways.

Science.gov (United States)

Li, Lan; Du, Yi; Ju, Furong; Ma, Shunxiang; Zhang, Shengxiang

2016-01-01

Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells. EL4 cells were exposed to POX for 0-16 h. EGTA was used to chelate Ca(2+ ) in extracellular medium, and heparin and procaine were used to inhibit Ca(2+ )efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis. POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78. Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.

Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells.

Science.gov (United States)

Qudrat, Anam; Truong, Kevin

2017-03-17

The versatility of Ca 2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca 2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca 2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca 2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca 2+ ). By coupling these chimeric proteins that generate Ca 2+ signals with proteins that respond to Ca 2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca 2+ . Thus, using this design strategy, it is possible to engineer proteins to generate a Ca 2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca 2+ -activated processes.

Experimental study of MRI signal changes of calcification

International Nuclear Information System (INIS)

Gong Xiangyang; Li Senhua; Li Rongfen; Hong Xiang; Gong Xiaoya; Xu Fengfeng

1999-01-01

Objective: To evaluate MRI signal changes according to different calcium compound, concentration and proportion, and try to give an reasonable explanation. Methods: Sixty samples composed of different calcium powders, various concentration and proportion of calcium were examined with CT and MRI. Five different calcium particles were evaluated with scanning electron microscopy. Results: (1) CT value of calcium gradually increased as the concentration increased; (2) CaSO 4 ·H 2 O was similar to CaCO 3 in terms of MRI T 1 WI signal intensity (P > 0.05); (3) Ca 3 (PO 4 ) 2 and Ca(OH) 2 showed hyperintensity in T 1 WI and was higher than other calcium salts (P 1 WI signal intensity of Ca 3 (PO 4 ) 2 / and Ca(OH) 2 showed biphasic curves with their peaks at 0.3 g/ml; (5) T 2 WI signal intensity of all series of calcium decreased as the concentration increased; (6) Signal intensity of mixed Ca 3 (PO 4 ) 2 /CaCO 3 was higher than CaHPO 4 ·2H 2 O/CaCO 3 on T 1 WI and lower on T 2 WI (P 3 , CaHPO 4 ·2H 2 O and CaSO 4 ·2H 2 O showed regular crystal shapes and smooth surface under scanning electron microscopy, but Ca 3 (PO 4 ) 2 and Ca(OH) 2 displayed their irregular figures and rough surface. Conclusions: Calcifications show variable MR signal due to difference of calcium compounds, various concentration and proportion of calcium. Understanding of these finding will help interpretation of MR images more precisely

Curcumin protects cortical neurons against oxygen and glucose deprivation/reoxygenation injury through flotillin-1 and extracellular signal-regulated kinase1/2 pathway.

Science.gov (United States)

Lu, Zhengyu; Liu, Yanping; Shi, Yang; Shi, Xinjie; Wang, Xin; Xu, Chuan; Zhao, Hong; Dong, Qiang

2018-02-05

In this study, we provided evidence that curcumin could be a promising therapeutic agent for ischemic stroke by activating neuroprotective signaling pathways. Post oxygen and glucose deprivation/reoxygenation (OGD/R), primary mouse cortical neurons treated with curcumin exhibited a significant decrease in cell death, LDH release and enzyme caspase-3 activity under OGD/R circumstances, which were abolished by flotillin-1 downregulation or extracellular signal-regulated kinase (ERK) inhibitor. Moreover, flotillin-1 knockdown led to suppression of curcumin-mediated ERK phosphorylation under OGD/R condition. Based on these findings, we concluded that curcumin could confer neuroprotection against OGD/R injury through a novel flotillin-1 and ERK1/2 pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling.

Science.gov (United States)

Feng, Wei; Kita, Daniel; Peaucelle, Alexis; Cartwright, Heather N; Doan, Vinh; Duan, Qiaohong; Liu, Ming-Che; Maman, Jacob; Steinhorst, Leonie; Schmitz-Thom, Ina; Yvon, Robert; Kudla, Jörg; Wu, Hen-Ming; Cheung, Alice Y; Dinneny, José R

2018-03-05

Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind di-glucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Guard Cell Signal Transduction Network: Advances in Understanding Abscisic Acid, CO2, and Ca2+ Signaling

KAUST Repository

Kim, Tae-Houn

2010-05-04

Stomatal pores are formed by pairs of specialized epidermal guard cells and serve as major gateways for both CO2 influx into plants from the atmosphere and transpirational water loss of plants. Because they regulate stomatal pore apertures via integration of both endogenous hormonal stimuli and environmental signals, guard cells have been highly developed as a model system to dissect the dynamics and mechanisms of plant-cell signaling. The stress hormone ABA and elevated levels of CO2 activate complex signaling pathways in guard cells that are mediated by kinases/phosphatases, secondary messengers, and ion channel regulation. Recent research in guard cells has led to a new hypothesis for how plants achieve specificity in intracellular calcium signaling: CO2 and ABA enhance (prime) the calcium sensitivity of downstream calcium-signaling mechanisms. Recent progress in identification of early stomatal signaling components are reviewed here, including ABA receptors and CO2-binding response proteins, as well as systems approaches that advance our understanding of guard cell-signaling mechanisms.

Guard Cell Signal Transduction Network: Advances in Understanding Abscisic Acid, CO2, and Ca2+ Signaling

KAUST Repository

Kim, Tae-Houn; Bö hmer, Maik; Hu, Honghong; Nishimura, Noriyuki; Schroeder, Julian I.

2010-01-01

Stomatal pores are formed by pairs of specialized epidermal guard cells and serve as major gateways for both CO2 influx into plants from the atmosphere and transpirational water loss of plants. Because they regulate stomatal pore apertures via integration of both endogenous hormonal stimuli and environmental signals, guard cells have been highly developed as a model system to dissect the dynamics and mechanisms of plant-cell signaling. The stress hormone ABA and elevated levels of CO2 activate complex signaling pathways in guard cells that are mediated by kinases/phosphatases, secondary messengers, and ion channel regulation. Recent research in guard cells has led to a new hypothesis for how plants achieve specificity in intracellular calcium signaling: CO2 and ABA enhance (prime) the calcium sensitivity of downstream calcium-signaling mechanisms. Recent progress in identification of early stomatal signaling components are reviewed here, including ABA receptors and CO2-binding response proteins, as well as systems approaches that advance our understanding of guard cell-signaling mechanisms.

Nanostructured gold microelectrodes for extracellular recording

Energy Technology Data Exchange (ETDEWEB)

Brueggemann, Dorothea; Wolfrum, Bernhard; Maybeck, Vanessa; Offenhaeusser, Andreas [CNI Center of Nanoelectronic Systems for Information Technology and Institute of Bio- and Nanosystems 2, Forschungszentrum Juelich (Germany)

2010-07-01

Electrophysiological activity of electrogenic cells is currently recorded with planar bioelectronic interfaces such as microelectrode arrays (MEAs). In this work, a novel concept of biocompatible nanostructured gold MEAs for extracellular signal recording is presented. MEAs were fabricated using clean room technologies, e.g. photolithography and metallization. Subsequently, they were modified with gold nanopillars of approximately 300 to 400 nm in height and 60 nm width. The nanostructuring process was carried out with a template-assisted approach using nanoporous aluminium oxide. Impedance spectroscopy of the resulting nanostructures showed higher capacitances compared to planar gold. This confirmed the expected increase of the surface area via nanostructuring. We used the nanostructured microelectrodes to record extracellular potentials from heart muscle cells (HL1), which were plated onto the chips. Good coupling between the HL1 cells and the nanostructured electrodes was observed. The resulting signal-to-noise ratio of nanopillar-MEAs was increased by a factor of 2 compared to planar MEAs. In future applications this nanopillar concept can be adopted for distinct interface materials and coupling to cellular and molecular sensing components.

Nuclear Calcium Buffering Capacity Shapes Neuronal Architecture.

Science.gov (United States)

Mauceri, Daniela; Hagenston, Anna M; Schramm, Kathrin; Weiss, Ursula; Bading, Hilmar

2015-09-18

Calcium-binding proteins (CaBPs) such as parvalbumin are part of the cellular calcium buffering system that determines intracellular calcium diffusion and influences the spatiotemporal dynamics of calcium signals. In neurons, CaBPs are primarily localized to the cytosol and function, for example, in nerve terminals in short-term synaptic plasticity. However, CaBPs are also expressed in the cell nucleus, suggesting that they modulate nuclear calcium signals, which are key regulators of neuronal gene expression. Here we show that the calcium buffering capacity of the cell nucleus in mouse hippocampal neurons regulates neuronal architecture by modulating the expression levels of VEGFD and the complement factor C1q-c, two nuclear calcium-regulated genes that control dendrite geometry and spine density, respectively. Increasing the levels of nuclear calcium buffers by means of expression of a nuclearly targeted form of parvalbumin fused to mCherry (PV.NLS-mC) led to a reduction in VEGFD expression and, as a result, to a decrease in total dendritic length and complexity. In contrast, mRNA levels of the synapse pruning factor C1q-c were increased in neurons expressing PV.NLS-mC, causing a reduction in the density and size of dendritic spines. Our results establish a close link between nuclear calcium buffering capacity and the transcription of genes that determine neuronal structure. They suggest that the development of cognitive deficits observed in neurological conditions associated with CaBP deregulation may reflect the loss of necessary structural features of dendrites and spines. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel calcium-sensing receptor cytoplasmic tail deletion mutation causing autosomal dominant hypocalcemia: molecular and clinical study.

Science.gov (United States)

Obermannova, Barbora; Sumnik, Zdenek; Dusatkova, Petra; Cinek, Ondrej; Grant, Michael; Lebl, Jan; Hendy, Geoffrey N

2016-04-01

Autosomal dominant hypocalcemia (ADH) is a rare disorder caused by activating mutations of the calcium-sensing receptor (CASR). The treatment of ADH patients with 1α-hydroxylated vitamin D derivatives can cause hypercalciuria leading to nephrocalcinosis. We studied a girl who presented with hypoparathyroidism and asymptomatic hypocalcemia at age 2.5 years. Mutations of CASR were investigated by DNA sequencing. Functional analyses of mutant and WT CASRs were done in transiently transfected human embryonic kidney (HEK293) cells. The proband and her father are heterozygous for an eight-nucleotide deletion c.2703_2710delCCTTGGAG in the CASR encoding the intracellular domain of the protein. Transient expression of CASR constructs in kidney cells in vitro suggested greater cell surface expression of the mutant receptor with a left-shifted extracellular calcium dose-response curve relative to that of the WT receptor consistent with gain of function. Initial treatment of the patient with calcitriol led to increased urinary calcium excretion. Evaluation for mosaicism in the paternal grandparents of the proband was negative. We describe a novel naturally occurring deletion mutation within the CASR that apparently arose de novo in the father of the ADH proband. Functional analysis suggests that the cytoplasmic tail of the CASR contains determinants that regulate the attenuation of signal transduction. Early molecular analysis of the CASR gene in patients with isolated idiopathic hypoparathyroidism is recommended because of its relevance to clinical outcome and treatment choice. In ADH patients, calcium supplementation and low-dose cholecalciferol avoids hypocalcemic symptoms without compromising renal function. © 2016 European Society of Endocrinology.

Excessive extracellular ATP desensitizes P2Y2 and P2X4 ATP receptors provoking surfactant impairment ending in ventilation-induced lung injury

NARCIS (Netherlands)

D. Hasan (Djo); Satalin, J. (Joshua); van der Zee, P. (Philip); Kollisch-Singule, M. (Michaela); P. Blankman (Paul); Shono, A. (Atsuko); P. Somhorst (Peter); C.A. den Uil (Corstiaan); H.J. Meeder (Han); Kotani, T. (Toru); G.F. Nieman (Gary F.)

2018-01-01

textabstractStretching the alveolar epithelial type I (AT I) cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP) (purinergic signaling). Extracellular ATP is cleared by extracellular ATPases,

Calcium regulation and Alzheimer’s disease

Directory of Open Access Journals (Sweden)

Deepthi Rapaka

2014-09-01

Full Text Available Activation of the neuron induces transient fluctuations in [Ca2+]i. This transient rise in [Ca2+]i is dependent on calcium entry via calcium channels and release of calcium from intracellular stores, finally resulting in increase in calcium levels, which activates calcium regulatory proteins to restore the resting calcium levels by binding to the calcium-binding proteins, sequestration into the endoplasmic reticulum and the mitochondria, and finally extrusion of calcium spike potential from the cell by adenosine triphosphate-driven Ca2+ pumps and the Na+/Ca2+ exchanger. Improper regulation of calcium signaling, sequentially, likely contributes to synaptic dysfunction and excitotoxic and/or apoptotic death of the vulnerable neuronal populations. The cognitive decline associated with normal aging is not only due to neuronal loss, but is fairly the result of synaptic connectivity. Many evidences support that Ca2+ dyshomeostasis is implicated in normal brain aging. Thus the chief factor associated with Alzheimer’s disease was found to be increase in the levels of free intracellular calcium, demonstrating that the excessive levels might lead to cell death, which provides a key target for the calcium channel blockers might be used as the neuroprotective agents in Alzheimer’s disease.

The alpha hemolisina of Escherichia Coli induces increases in the calcium citoplasmico of neutrofilos and monocytes human beings

International Nuclear Information System (INIS)

Garcia, J.

2000-01-01

Escherichia coli alpha hemolysin (AH) and the calcium ionophores ionomycin and 4 Br A23187 caused increases in cell fluorescence, indicative of elevations in cytoplasmic calcium, in fura 2-loaded human polymorphonuclear leukocytes(PMN) and monocytes (MN). The increase in fluorescence caused by AH was dose dependent. Quelation of extracellular calcium with EGTA prevented fluorescence increases in PMN exposed to 2 HU50/ml AH, but did not prevent a small increase in 4 μM, ionomycin-treated PMN, indicating that ionomycin treatment under conditions of calcium quelation can mobilize calcium from internal stores, and that entry of external calcium accounts for most of the increases in cell fluorescence in cells treated with both AH and calcium ionophores. AH, as well as calcium ionophores and the chemotactic peptide FMLP caused rease of myeloperoxidase (MPO) from PMM suggesting that increments in intracellular calcium cause degramulation with release of granule contents (Author) [es

Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

Directory of Open Access Journals (Sweden)

Wagner Shin Nishitani

Full Text Available A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7 expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

In vivo experimental stroke and in vitro organ culture induce similar changes in vasoconstrictor receptors and intracellular calcium handling in rat cerebral arteries

DEFF Research Database (Denmark)

Povlsen, Gro Klitgaard; Waldsee, Roya; Ahnstedt, Hilda

2012-01-01

Cerebral arteries subjected to different types of experimental stroke upregulate their expression of certain G-protein-coupled vasoconstrictor receptors, a phenomenon that worsens the ischemic brain damage. Upregulation of contractile endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B......)) receptors has been demonstrated after subarachnoid hemorrhage and global ischemic stroke, but the situation is less clear after focal ischemic stroke. Changes in smooth muscle calcium handling have been implicated in different vascular diseases but have not hitherto been investigated in cerebral arteries...... and extracellular sources, whereas 5-HT(1B) receptor-mediated contraction was solely dependent on extracellular calcium. Organ culture and stroke increased basal intracellular calcium levels in MCA smooth muscle cells and decreased the expression of inositol triphosphate receptor and transient receptor potential...

Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

Energy Technology Data Exchange (ETDEWEB)

Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

2014-11-15

New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

International Nuclear Information System (INIS)

Voss, Kelsey; Amaya, Moushimi; Mueller, Claudius; Roberts, Brian; Kehn-Hall, Kylene; Bailey, Charles; Petricoin, Emanuel; Narayanan, Aarthi

2014-01-01

New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells

Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress.

Science.gov (United States)

Aslam, Roohi; Williams, Lorraine E; Bhatti, Muhammad Faraz; Virk, Nasar

2017-10-27

P 2 - type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca 2+ , Mn 2+ and Zn 2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat. In this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P 2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P 2- type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat. Here we concluded that wheat genome consists of nine P 2B and three P 2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P 2A and P 2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be

Shaping Synapses by the Neural Extracellular Matrix

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Maura Ferrer-Ferrer

2018-05-01

Full Text Available Accumulating data support the importance of interactions between pre- and postsynaptic neuronal elements with astroglial processes and extracellular matrix (ECM for formation and plasticity of chemical synapses, and thus validate the concept of a tetrapartite synapse. Here we outline the major mechanisms driving: (i synaptogenesis by secreted extracellular scaffolding molecules, like thrombospondins (TSPs, neuronal pentraxins (NPs and cerebellins, which respectively promote presynaptic, postsynaptic differentiation or both; (ii maturation of synapses via reelin and integrin ligands-mediated signaling; and (iii regulation of synaptic plasticity by ECM-dependent control of induction and consolidation of new synaptic configurations. Particularly, we focused on potential importance of activity-dependent concerted activation of multiple extracellular proteases, such as ADAMTS4/5/15, MMP9 and neurotrypsin, for permissive and instructive events in synaptic remodeling through localized degradation of perisynaptic ECM and generation of proteolytic fragments as inducers of synaptic plasticity.

Stress-induced ECM alteration modulates cellular microRNAs that feedback to readjust the extracellular environment and cell behaviour

Directory of Open Access Journals (Sweden)

Halyna R Shcherbata

2013-12-01

Full Text Available The extracellular environment is a complex entity comprising of the extracellular matrix (ECM and regulatory molecules. It is highly dynamic and under cell-extrinsic stress, transmits the stressed organism’s state to each individual ECM-connected cell. microRNAs (miRNAs are regulatory molecules involved in virtually all the processes in the cell, especially under stress. In this review, we analyse how microRNA expression is regulated downstream of various signal transduction pathways induced by changes in the extracellular environment. In particular, we focus on the muscular dystrophy-associated cell adhesion molecule dystroglycan capable of signal transduction. Then we show how exactly the same miRNAs feedback to regulate the extracellular environment. The ultimate goal of this bi-directional signal transduction process is to change cell behaviour under cell-extrinsic stress in order to respond to it accordingly.

Calcium and Bone Metabolism Indices.

Science.gov (United States)

Song, Lu

2017-01-01

Calcium and inorganic phosphate are of critical importance for many body functions, thus the regulations of their plasma concentrations are tightly controlled by the concerted actions of reabsorption/excretion in the kidney, absorption in the intestines, and exchange from bone, the major reservoir for calcium and phosphate in the body. Parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH) 2 D) control calcium homeostasis, whereas PTH, 1,25(OH) 2 D, and bone-derived fibroblast growth factor 23 (FGF 23) control phosphate homeostasis. Hypoparathyroidism can cause hypocalcemia and hyperphosphatemia, whereas deficient vitamin D actions can cause osteomalacia in adults and rickets in children. Hyperparathyroidism, alternatively, can cause hypercalcemia and hypophosphatemia. Laboratory tests of calcium, phosphate, PTH, and 25-hydroxyvitamin D are very useful in the diagnosis of abnormalities associated with calcium and/or phosphate metabolisms. Bone is constantly remodeled throughout life in response to mechanical stress and a need for calcium in extracellular fluids. Metabolic bone diseases such as osteoporosis, osteomalacia in adults or rickets in children, and renal osteodystrophy develop when bone resorption exceeds bone formation. Bone turnover markers (BTM) such as serum N-terminal propeptide of type I procollagen (P1NP) and C-terminal collagen cross-link (CTX) may be useful in predicting future fracture risk or monitoring the response to anti-resorptive therapy. There is a need to standardize sample collection protocols because certain BTMs exhibit large circadian variations and tend to be influenced by food intakes. In the United States, a project to standardize BTM sample collection protocols and to establish the reference intervals for serum P1NP and serum CTX is ongoing. We anticipate the outcome of this project to shine lights on the standardization of BTM assays, sample collection protocols, reference intervals in relation to age, sex, and ethnic

Zinc release in the lateral nucleus of the amygdala by stimulation of the entorhinal cortex.

Science.gov (United States)

Takeda, Atsushi; Imano, Sachie; Itoh, Hiromasa; Oku, Naoto

2006-11-06

Zinc release in the lateral nucleus of the amygdala was examined using rat brain slices. The lateral and basolateral nuclei in the amygdala were evidently stained by Timm's sulfide-silver staining method. When the amygdala including both the nuclei was stimulated with 100 mM KCl by means of in vivo microdialysis, extracellular zinc concentration was increased significantly. Zinc release in the lateral nucleus of the amygdala innervated by the entorhinal cortex was next examined in brain slices double-stained with zinc and calcium indicators. Extracellular zinc signal (ZnAF-2) in the lateral nucleus was increased with intracellular calcium signal (calcium orange) during delivery of tetanic stimuli to the entorhinal cortex. Both the increases were completely inhibited by addition of 1 micro M tetrodotoxin, a sodium channel blocker. Furthermore, calcium signal in the lateral nucleus during delivery of tetanic stimuli to the entorhinal cortex was increased in the presence of 10 micro M CNQX, an AMPA/KA receptor antagonist, and this increase was facilitated by addition of 1 mM CaEDTA, a membrane-impermeable zinc chelator. The present study suggested that zinc is released in the lateral nucleus of the amygdala by depolarization of the entorhinal neurons. In the lateral nucleus, zinc released may suppress the increase in presynaptic calcium signal.

Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

International Nuclear Information System (INIS)

Hillmann, Petra; Koese, Meryem; Soehl, Kristina; Mueller, Christa E.

2008-01-01

High blood levels of ammonium/ammonia (NH 4 + /NH 3 ) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca 2+ ] i ) in astrocytoma (EC 50 = 6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca 2+ ] i was due to an intracellular effect of NH 4 + /NH 3 and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by G q protein-coupled receptors (H 1 and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH 3 /NH 4 + from neuronal cells. Cellular uptake of NH 4 + /NH 3 was mainly by passive diffusion of NH 3 . Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia

Chaotic Dynamics Mediates Brain State Transitions, Driven by Changes in Extracellular Ion Concentrations

DEFF Research Database (Denmark)

Rasmussen, Rune; H. Jensen, Mogens; L. Heltberg, Mathias

2017-01-01

Previous studies have suggested that changes in extracellular ion concentrations initiate the transition from an activity state that characterizes sleep in cortical neurons to states that characterize wakeful- ness. However, because neuronal activity and extra- cellular ion concentrations...... are interdependent, isolating their unique roles during sleep-wake transitions is not possible in vivo. Here, we extend the Averaged-Neuron model and demonstrate that, although changes in extracellular ion concentrations occur concurrently, decreasing the conductance of calcium-dependent potassium channels initiates...... the transition from sleep to wakefulness. We find that sleep is governed by stable, self-sustained oscillations in neuronal firing patterns, whereas the quiet awake state and active awake state are both governed by irregular oscillations and chaotic dynamics; transitions between these separable awake states...

Glucose: an Energy Currency and Structural Precursor in Articular Cartilage and Bone with Emerging Roles as an Extracellular Signalling Molecule and Metabolic Regulator

Directory of Open Access Journals (Sweden)

Ali eMobasheri

2012-12-01

Full Text Available In the musculoskeletal system glucose serves as an essential source of energy for the development, growth and maintenance of bone and articular cartilage. It is particularly needed for skeletal morphogenesis during embryonic growth and foetal development. Glucose is vital for osteogenesis and chondrogenesis, and is used as a precursor for the synthesis of glycosaminoglycans, glycoproteins and glycolipids. Glucose sensors are present in tissues and organs that carry out bulk glucose fluxes (i.e. intestine, kidney and liver. The beta cells of the pancreatic islets of Langerhans respond to changes in glucose concentration by varying the rate of insulin synthesis and secretion. Neuronal cells in the hypothalamus are also capable of sensing extracellular glucose. Glucosensing neurons use glucose as a signalling molecule to alter their action potential frequency in response to variations in ambient glucose levels. Skeletal muscle and adipose tissue can respond to changes in circulating glucose but much less is known about glucosensing in bone and cartilage. Recent research suggests that bone cells can influence (and be influenced by systemic glucose metabolism. This focused review article discusses what we know about glucose transport and metabolism in bone and cartilage and highlights recent studies that have linked glucose metabolism, insulin signalling and osteocalcin activity in bone and cartilage. These new findings in bone cells raise important questions about nutrient sensing, uptake, storage and processing mechanisms and how they might contribute to overall energy homeostasis in health and disease. The role of glucose in modulating anabolic and catabolic gene expression in normal and osteoarthritic chondrocytes is also discussed. In summary, cartilage and bone cells are sensitive to extracellular glucose and adjust their gene expression and metabolism in response to varying extracellular glucose concentrations.

The control of calcium signaling in the heart

African Journals Online (AJOL)

Dr Olaleye Samuel

130 years later one can still admire the mind that, having discovered that the previous year's result was not reproducible, realized that the problem was due to a contaminant and then correctly identified calcium as the culprit. Several other things are worth noting in this anecdote. (1) Ringer's exhaustive work provided the.

Identification of a receptor for extracellular renalase.

Directory of Open Access Journals (Sweden)

Ling Wang

Full Text Available An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI in wild type (WT mice. Therefore, we sought to identity the receptor for extracellular renalase.RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

Science.gov (United States)

Kashir, Junaid; Deguchi, Ryusaku; Jones, Celine; Coward, Kevin; Stricker, Stephen A

2013-10-01

Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca²⁺) in all animals that have been examined, and such Ca²⁺ elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca²⁺ transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad-based analysis of Ca²⁺ dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non-mammalian species, and subsequently reviews various topics related to a sperm-specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca²⁺ signals in fertilized oocytes or eggs are compared in a taxon-by-taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization-induced Ca²⁺ signals are typically propagated as global waves that depend on Ca²⁺ release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5-trisphosphate (IP₃). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less-common modes of producing Ca²⁺ transients are also examined. In addition, the importance of fertilization-induced Ca²⁺ signals for activating development is underscored by noting some major downstream effects of these signals in various animals. © 2013 Wiley Periodicals, Inc.

Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin

Science.gov (United States)

Yang, T.; Poovaiah, B. W.

2002-01-01

Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome.

Science.gov (United States)

Doyle, Jefferson J; Doyle, Alexander J; Wilson, Nicole K; Habashi, Jennifer P; Bedja, Djahida; Whitworth, Ryan E; Lindsay, Mark E; Schoenhoff, Florian; Myers, Loretha; Huso, Nick; Bachir, Suha; Squires, Oliver; Rusholme, Benjamin; Ehsan, Hamid; Huso, David; Thomas, Craig J; Caulfield, Mark J; Van Eyk, Jennifer E; Judge, Daniel P; Dietz, Harry C

2015-10-27

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCβ) as a critical mediator of this pathway and demonstrate that the PKCβ inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCβ and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.

Neuronal extracellular signal-regulated kinase (ERK activity as marker and mediator of alcohol and opioid dependence

Directory of Open Access Journals (Sweden)

Eva R. Zamora-Martinez

2014-03-01

Full Text Available Early pioneering work in the field of biochemistry identified phosphorylation as a crucial post-translational modification of proteins with the ability to both indicate and arbitrate complex physiological processes. More recent investigations have functionally linked phosphorylation of extracellular signal-regulated kinase (ERK to a variety of neurophysiological mechanisms ranging from acute neurotransmitter action to long-term gene expression. ERK phosphorylation serves as an intracellular bridging mechanism that facilitates neuronal communication and plasticity. Drugs of abuse, including alcohol and opioids, act as artificial yet powerful rewards that impinge upon natural reinforcement processes critical for survival. The graded progression from initial exposure to addiction (or substance dependence is believed to result from drug- and drug context-induced adaptations in neuronal signaling processes across brain reward and stress circuits following excessive drug use. In this regard, commonly abused drugs as well as drug-associated experiences are capable of modifying the phosphorylation of ERK within central reinforcement systems. In addition, chronic drug and alcohol exposure may drive ERK-regulated epigenetic and structural alterations that underlie a long-term propensity for escalating drug use. Under the influence of such a neurobiological vulnerability, encountering drug-associated cues and contexts can produce subsequent alterations in ERK signaling that drive relapse to drug and alcohol seeking. Current studies are determining precisely which molecular and regional ERK phosphorylation-associated events contribute to the addiction process, as well as which neuroadaptations need to be targeted in order to return dependent individuals to a healthy state.

Electric field stimulation through a substrate influences Schwann cell and extracellular matrix structure

Science.gov (United States)

Nguyen, Hieu T.; Wei, Claudia; Chow, Jacqueline K.; Nguy, Lindsey; Nguyen, Hieu K.; Schmidt, Christine E.

2013-08-01

Objective. Electric field (EF) stimulation has been used to cue cell growth for tissue engineering applications. In this study, we explore the electrical parameters and extracellular mechanisms that elicit changes in cell behavior when stimulated through the substrate. Approach. Rat Schwann cell morphology was compared when exposed to EF through the media or a conductive indium tin oxide substrate. Ionic and structural effects were then analyzed on Matrigel and collagen I, respectively. Main results. When stimulating through media, cells had greater alignment perpendicular to the EF with higher current densities (106 mA cm-2 at 245 mV mm-1), and reached maximum alignment within 8 h. Stimulation through the substrate with EF (up to 110 mV mm-1) did not affect Schwann cell orientation, however the EF caused extracellular matrix (ECM) coatings on substrates to peel away, suggesting EF can physically change the ECM. Applying alternating current (ac) 2-1000 Hz signals through the media or substrate both caused cells to flatten and protrude many processes, without preferential alignment. Matrigel exposed to a substrate EF of 10 mV mm-1 for 2 h had a greater calcium concentration near the cathode, but quickly dissipated when the EF was removed. Schwann cells seeded 7 d after gels were exposed to substrate EF still aligned perpendicular to the EF direction. Microscopy of collagen I exposed to substrate EF shows alignment and bundling of fibrils. Significance. These findings demonstrate EF exposure can control Schwann cell alignment and morphology, change ECM bulk/surface architecture, and align ECM structures.

Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein.

Science.gov (United States)

Tao, Shi-Cong; Gao, You-Shui; Zhu, Hong-Yi; Yin, Jun-Hui; Chen, Yi-Xuan; Zhang, Yue-Lei; Guo, Shang-Chun; Zhang, Chang-Qing

2016-06-03

The pH of extracellular fluids is a basic property of the tissue microenvironment and is normally maintained at 7.40 ± 0.05 in humans. Many pathological circumstances, such as ischemia, inflammation, and tumorigenesis, result in the reduction of extracellular pH in the affected tissues. In this study, we reported that the osteogenic differentiation of BMSCs was significantly inhibited by decreases in the extracellular pH. Moreover, we demonstrated that proton-sensing GPR4 signaling mediated the proton-induced inhibitory effects on the osteogenesis of BMSCs. Additionally, we found that YAP was the downstream effector of GPR4 signaling. Our findings revealed that the extracellular pH modulates the osteogenic responses of BMSCs by regulating the proton-sensing GPR4-YAP pathway.

Effect of free calcium concentration and ionic strength on alginate fouling in cross-flow membrane filtration

NARCIS (Netherlands)

Brink, van den P.; Zwijnenburg, A.; Smith, G.; Temmink, B.G.; Loosdrecht, van M.C.

2009-01-01

Extracellular polymeric substances (EPS) are generally negatively charged polymers. Membrane fouling in membrane bioreactors (MBRs) by EPS is therefore influenced by the water chemistry of the mixed liquor (calcium concentration, foulant concentration and ionic strength). We used alginate as a model

The role of Rho-kinase and calcium ions in constriction triggered by ET-1.

Science.gov (United States)

Wiciński, Michał; Szadujkis-Szadurska, Katarzyna; Węclewicz, Mateusz M; Malinowski, Bartosz; Matusiak, Grzegorz; Walczak, Maciej; Wódkiewicz, Eryk; Grześk, Grzegorz; Pawlak-Osińska, Katarzyna

2018-05-05

Endothelin-1 (ET-1) is one of the key factors regulating tension of smooth muscles in blood vessels. It is believed that ET-1 plays an important role in pathogenesis of hypertension, and cardiovascular diseases; therefore, research in order to limit ET-1-mediated action is still in progress. The main objective of this paper was to evaluate the role of Rho-kinase in the ET-1-induced constriction of arteries. The analysis also included significance of intra- and extracellular pool of calcium ions in constriction triggered by ET-1. The studies were performed on perfused Wistar rat tail arteries. Concentration response curve (CRC) was determined for ET-1 in the presence of increased concentrations of Rho-kinase inhibitor (Y-27632) and IP3-receptor antagonist (2APB), both in reference to constriction triggered by solely ET-1. Afterwards, the influence of calcium ions present in the perfusion fluid was evaluated in terms of the effect triggered by 2APB and occurring in arteries constricted by ET-1. ET-1, in concentration dependent manner, leads to increase in perfusion pressure. Y-27632 and 2APB lead to shift of the concentration response curve for ET-1 to the right with simultaneously lowered maximum effect. There was no difference in reaction of the artery constricted by ET-1 and treated with 2APB in solution containing calcium and in calcium-free solution. Vasoconstrictive action of endothelin is not significantly dependent on the inflow of extracellular calcium, but it is proportional to inflow of Ca 2+ related to activation of IP3 receptors and to Rho-kinase activity. Copyright © 2018. Published by Elsevier Inc.

Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

Science.gov (United States)

Contreras, Laura; Satrústegui, Jorgina

2009-03-13

Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

Membrane mechanisms and intracellular signalling in cell volume regulation

DEFF Research Database (Denmark)

Hoffmann, Else Kay; Dunham, Philip B.

1995-01-01

Volume regulation, Signal transduction, Calcium-calmodulin, Stretch-activated channels, Eicosanoids, Macromolecular crowding, Cytoskeleton, Protein phosphorylation, dephosphorylation.......Volume regulation, Signal transduction, Calcium-calmodulin, Stretch-activated channels, Eicosanoids, Macromolecular crowding, Cytoskeleton, Protein phosphorylation, dephosphorylation....

Odontogenic differentiation of human dental pulp cells by calcium silicate materials stimulating via FGFR/ERK signaling pathway

International Nuclear Information System (INIS)

Liu, Chao-Hsin; Hung, Chi-Jr; Huang, Tsui-Hsien; Lin, Chi-Chang; Kao, Chia-Tze; Shie, Ming-You

2014-01-01

Bone healing needs a complex interaction of growth factors that establishes an environment for efficient bone formation. We examine how calcium silicate (CS) and tricalcium phosphate (β-TCP) cements influence the behavior of human dental pulp cells (hDPCs) through fibroblast growth factor receptor (FGFR) and active MAPK pathways, in particular ERK. The hDPCs are cultured with β-TCP and CS, after which the cells' viability and odontogenic differentiation markers are determined by using PrestoBlue® assay and western blot, respectively. The effect of small interfering RNA (siRNA) transfection targeting FGFR was also evaluated. The results showed that CS promoted cell proliferation and enhances FGFR expression. It was also found that CS increases ERK and p38 activity in hDPCs, and furthermore, raises the expression and secretion of DSP, and DMP-1. Additionally, statistically significant differences (p < 0.05) have been found in the calcium deposition in si-FGFR transfection and ERK inhibitor between CS and β-TCP; these variations indicated that ERK/MAPK signaling is involved in the silicon-induced odontogenic differentiation of hDPCs. The current study shows that CS substrates play a key role in odontoblastic differentiation of hDPCs through FGFR and modulate ERK/MAPK activation. - Highlights: • CS influences the behavior of hDPCs through fibroblast growth factor receptor. • CS increases ERK and p38 activity in hDPCs. • ERK/MAPK signaling is involved in the Si-induced odontogenic differentiation of hDPCs. • Ca staining shows that FGFR regulates hDPC differentiation on CS, but not on β-TCP

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.

Science.gov (United States)

Eigenheer, Richard A; Jin Lee, Young; Blumwald, Eduardo; Phinney, Brett S; Gelli, Angie

2007-06-01

Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.

Hydrogen sulfide interacts with calcium signaling to enhance the chromium tolerance in Setaria italica.

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Fang, Huihui; Jing, Tao; Liu, Zhiqiang; Zhang, Liping; Jin, Zhuping; Pei, Yanxi

2014-12-01

The oscillation of intracellular calcium (Ca(2+)) concentration is a primary event in numerous biological processes in plants, including stress response. Hydrogen sulfide (H2S), an emerging gasotransmitter, was found to have positive effects in plants responding to chromium (Cr(6+)) stress through interacting with Ca(2+) signaling. While Ca(2+) resemblances H2S in mediating biotic and abiotic stresses, crosstalk between the two pathways remains unclear. In this study, Ca(2+) signaling interacted with H2S to produce a complex physiological response, which enhanced the Cr(6+) tolerance in foxtail millet (Setaria italica). Results indicate that Cr(6+) stress activated endogenous H2S synthesis as well as Ca(2+) signaling. Moreover, toxic symptoms caused by Cr(6+) stress were strongly moderated by 50μM H2S and 20mM Ca(2+). Conversely, treatments with H2S synthesis inhibitor and Ca(2+) chelators prior to Cr(6+)-exposure aggravated these toxic symptoms. Interestingly, Ca(2+) upregulated expression of two important factors in metal metabolism, MT3A and PCS, which participated in the biosynthesis of heavy metal chelators, in a H2S-dependent manner to cope with Cr(6+) stress. These findings also suggest that the H2S dependent pathway is a component of the Ca(2+) activating antioxidant system and H2S partially contributes Ca(2+)-activating antioxidant system. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

Science.gov (United States)

Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

2014-11-01

The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx

DEFF Research Database (Denmark)

Amstrup, Jan; Novak, Ivana

2003-01-01

P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study...... we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized...... to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor...

The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

Directory of Open Access Journals (Sweden)

Yajin Liao

2017-02-01

Full Text Available The mitochondrial calcium uniporter (MCU—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP; however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders.

Parallel Stochastic discrete event simulation of calcium dynamics in neuron.

Science.gov (United States)

Ishlam Patoary, Mohammad Nazrul; Tropper, Carl; McDougal, Robert A; Zhongwei, Lin; Lytton, William W

2017-09-26

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 its concentration (percentage-wise). These rare events can affect dynamics discretely in such way that they cannot be evaluated by a deterministic 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 serial deterministic simulation in NEURON. We also derived a discrete event calcium wave model from a deterministic model using the stochastic IP3R structure.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

Science.gov (United States)

Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

2015-01-01

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

Calcium Signals from the Vacuole

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Gerald Schönknecht

2013-10-01

Full Text Available The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.

T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

DEFF Research Database (Denmark)

Poulsen, Christian B; Al-Mashhadi, Rozh H; Cribbs, Leanne L

2011-01-01

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar...... tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium...... by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response...

Zinc signaling in the hippocampus and its relation to pathogenesis of depression.

Science.gov (United States)

Takeda, Atsushi

2012-06-01

Histochemically reactive zinc (Zn(2+)) is co-released with glutamate from zincergic neurons, a subclass of glutamatergic neurons. Zn(2+) serves as a signal factor in both the extracellular and intracellular compartments. Glucocorticoid-glutamatergic interactions have been proposed as a potential model to explain stress-mediated impairment of hippocampal function, i.e., cognition. However, it is unknown whether glucocorticoid-zincergic interactions are involved in this impairment. In the present study, involvement of synaptic Zn(2+) in stress-induced attenuation of CA1 LTP was examined in hippocampal slices from young rats after exposure to tail suspension stress for 30s, which significantly increased serum corticosterone. Stress-induced attenuation of CA1 LTP was ameliorated by administration of clioquinol, a membrane permeable zinc chelator, to rats prior to exposure to stress, implying that the reduction of synaptic Zn(2+) by clioquinol participates in this amelioration. To pursue the involvement of corticosterone-mediated Zn(2+) signal in the attenuated CA1 LTP by stress, dynamics of synaptic Zn(2+) was checked in hippocampal slices exposed to corticosterone. Corticosterone increased extracellular Zn(2+) levels measured with ZnAF-2 dose-dependently, as well as the intracellular Ca(2+) levels measured with calcium orange AM, suggesting that corticosterone excites zincergic neurons in the hippocampus and increases Zn(2+) release from the neuron terminals. Intracellular Zn(2+) levels measured with ZnAF-2DA were also increased dose-dependently, but not in the coexistence of CaEDTA, a membrane-impermeable zinc chelator, suggesting that intracellular Zn(2+) levels is increased by the influx of extracellular Zn(2+). Furthermore, corticosterone-induced attenuation of CA1 LTP was abolished in the coexistence of CaEDTA. The present study suggests that corticosterone-mediated increase in postsynaptic Zn(2+) signal in the cytosolic compartment is involved in the attenuation

Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells.

Science.gov (United States)

Lecourieux, David; Mazars, Christian; Pauly, Nicolas; Ranjeva, Raoul; Pugin, Alain

2002-10-01

Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca(2+)](cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration. The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration. Cryptogein signature is characterized by a long-sustained [Ca(2+)](cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death. The [Ca(2+)](cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx. H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in [Ca(2+)](cyt) increase and may activate calcium channels from the plasma membrane. Competition assays with different elicitins demonstrate that [Ca(2+)](cyt) increase is mediated by cryptogein-receptor interaction.

Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

Science.gov (United States)

Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

2016-01-01

Rice ( Oryza sativa ) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species

Ouabain enhancement of compound 48/80 induced histamine secretion from rat peritoneal mast cells: dependence on extracellular sodium

DEFF Research Database (Denmark)

Knudsen, T; Bertelsen, Niels Haldor; Johansen, Torben

1992-01-01

Purified populations of rat peritoneal mast cells were used to study the effect of ouabain on compound 48/80-induced histamine secretion and on 86Rb+ uptake. 86Rb+ was used as a tracer for extracellular K+. The calculated value of the ouabain-sensitive uptake of K+ and 86Rb+ was considered...... on the secretion occurs in the presence of sodium but not when sodium was replaced by lithium. Preservation by ouabain of a high intracellular sodium content in sodium-loaded cells was associated with preservation of the secretory response in a calcium-free medium. In the presence of lanthanum in a calcium...

Visualisation of an nsPEF induced calcium wave using the genetically encoded calcium indicator GCaMP in U87 human glioblastoma cells.

Science.gov (United States)

Carr, Lynn; Bardet, Sylvia M; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

2018-02-01

Cytosolic, synthetic chemical calcium indicators are typically used to visualise the rapid increase in intracellular calcium ion concentration that follows nanosecond pulsed electric field (nsPEF) application. This study looks at the application of genetically encoded calcium indicators (GECIs) to investigate the spatiotemporal nature of nsPEF-induced calcium signals using fluorescent live cell imaging. Calcium responses to 44kV/cm, 10ns pulses were observed in U87-MG cells expressing either a plasma membrane targeted GECI (GCaMP5-G), or one cytosolically expressed (GCaMP6-S), and compared to the response of cells loaded with cytosolic or plasma membrane targeted chemical calcium indicators. Application of 100 pulses, to cells containing plasma membrane targeted indicators, revealed a wave of calcium across the cell initiating at the cathode side. A similar spatial wave was not observed with cytosolic indicators with mobile calcium buffering properties. The speed of the wave was related to pulse application frequency and it was not propagated by calcium induced calcium release. Copyright © 2017 Elsevier B.V. All rights reserved.

Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

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John Paul Christianson

2014-10-01

Full Text Available Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed behavioral immunization. Recent data implicate neuroplasticity within the ventromedial prefrontal cortex (mPFC as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and one week later to uncontrollable tailshocks, followed 24h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDAR and the extracellular signal-regulated kinase (ERK cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL or infralimbic (IL mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent plasticity within the PL region is required for behavioral immunization, a learned form of stressor resistance.

Intracellular calcium homeostasis and signaling.

Science.gov (United States)

Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

2013-01-01

Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

Calcium measurements in living filamentous fungi expressing codon-optimized aequorin

NARCIS (Netherlands)

Nelson, G.; Kozlova-Zwinderman, O.; Collis, A.J.; Knight, M.R.; Fincham, J.R.S.; Stanger, C.P.; Renwick, A.; Hessing, J.G.M.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Read, N.D.

2004-01-01

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in

STIM and Orai isoform expression in pregnant human myometrium: a potential role in calcium signaling during pregnancy.

Directory of Open Access Journals (Sweden)

Evonne eChin-Smith

2014-05-01

Full Text Available Store-operated calcium (Ca2+ entry (SOCE can be mediated by two novel proteins, STIM/Orai. We have previously demonstrated that members of the TRPC family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labor associated stimuli IL-1β and mechanical stretch. Although STIM and Orai isoforms (1-3 have been reported in other smooth muscle cell types, there is little known about the expression or gestational regulation of STIM and Orai expression in human myometrium. Total RNA was isolated from lower segment human myometrial biopsies obtained at caesarean section from women at the time of preterm no labor (PTNL, preterm labor (PTL, term non-labor (TNL and term with labor (TL; primary cultured human uterine smooth muscle cells, and a human myometrial cell line (hTERT-HM. STIM1-2, and Orai1-3 mRNA expression was assessed by quantitative real-time PCR. All five genes were expressed in myometrial tissue and cultured cells. Orai2 was the most abundant Orai isoform in human myometrium. Expression of STIM1-2/Orai1-3 did not alter with the onset of labor. Orai1 mRNA expression in cultured cells was enhanced by IL-1β treatment. This novel report of STIM1-2 and Orai1-3 mRNA expression in pregnant human myometrium and Orai1 regulation by IL-1β indicates a potential role for these proteins in calcium signaling in human myometrium during pregnancy.

Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells.

Science.gov (United States)

Misonou, Yoshiko; Takahashi, Motoko; Park, Yong Seek; Asahi, Michio; Miyamoto, Yasuhide; Sakiyama, Haruhiko; Cheng, Xinyao; Taniguchi, Naoyuki

2005-05-01

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.

Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region.

Science.gov (United States)

Grzesik, Paul; Kreuchwig, Annika; Rutz, Claudia; Furkert, Jens; Wiesner, Burkhard; Schuelein, Ralf; Kleinau, Gunnar; Gromoll, Joerg; Krause, Gerd

2015-01-01

The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

International Nuclear Information System (INIS)

Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias; Malmberg, Karl-Johan; Troye-Blomberg, Marita; Ahlborg, Niklas; Karlsson, Anna; Johansson, Magnus; Lundberg, Mathias

2009-01-01

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.

Diverse roles of extracellular calcium-sensing receptor in the central nervous system

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Bandyopadhyay, Sanghamitra; Tfelt-Hansen, Jacob; Chattopadhyay, Naibedya

2010-01-01

The G-protein-coupled calcium-sensing receptor (CaSR), upon activation by Ca(2+) or other physiologically relevant polycationic molecules, performs diverse functions in the brain. The CaSR is widely expressed in the central nervous system (CNS) and is characterized by a robust increase in its...... to astrocytic or neuronal lineages. In adult CNS, CaSR has broad relevance in maintaining local ionic homeostasis. CaSR shares an evolutionary relationship with the metabotropic glutamate receptor and forms heteromeric complexes with the type B-aminobutyric acid receptor subunits that affects its cell surface...

Extracellular vesicles provide a means for tissue crosstalk during exercise

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Whitham, Martin; Parker, Benjamin L; Friedrichsen, Martin

2018-01-01

Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative prot...

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

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De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells.

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Maeda, Toyonobu; Suzuki, Atsuko; Koga, Kaori; Miyamoto, Chihiro; Maehata, Yojiro; Ozawa, Shigeyuki; Hata, Ryu-Ichiro; Nagashima, Yoji; Nabeshima, Kazuki; Miyazaki, Kaoru; Kato, Yasumasa

2017-10-03

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca 2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca 2+ -dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

Syndecans as receptors and organizers of the extracellular matrix.

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Xian, Xiaojie; Gopal, Sandeep; Couchman, John R

2010-01-01

Syndecans are type I transmembrane proteins having a core protein modified with glycosaminoglycan chains, most commonly heparan sulphate. They are an ancient group of molecules, present in invertebrates and vertebrates. Among the plethora of molecules that can interact with heparan sulphate, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles in postnatal tissue repair, inflammation and tumour progression. Developmental deficits in lower vertebrates in which syndecans are eliminated are also informative and suggest that, in mammals, redundancy is a key issue.

Extracellular sphingosine-1-phosphate: a novel actor in human glioblastoma stem cell survival.

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Elena Riccitelli

Full Text Available Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs, a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ, and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source

Comparison of radioisotopic studied calcium metabolism in the orally administered and inhaled cadmium rat

International Nuclear Information System (INIS)

Fauran-Clavel, M.J.; Oustrin, J.; Godin, J.; Boudene, C.

1982-01-01

The radioisotopic study of calcium metabolism in the rat after oral administration of cadmium, 8 mg/kg during 13 weeks, has shown two different effects of this ion: 1) in the intestine, cadmium inhibits the absorption of calcium by active transport; 2) in the deep bone compartment, the decrease of the bone calcium used for the crystallization and slowly exchangeable with the calcium central pool (serum, extracellular and soft tissues calcium) is combined with a reduction of the exchange rates between the two compartments. When administered through a microparticle aerosol inhalation (1 mg/m 3 of air, 30 mn a day, during 12 weeks), cadmium's main target organ is the deep bone compartment. For both modes of administration, the slowing down of osteogenesis is confirmed by a drop in serum alkaline phosphatase after a four weeks period which reflects a decrease of the osteoblastic activity. Therefore it appears that the effects on bones observed during the chronic oral cadmium administration, do not result from a malabsorption of intestine calcium but also from the very action the Cd ++ ion on the bone crystallization process [fr

Inflammation leads to distinct populations of extracellular vesicles from microglia

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Yang, Yiyi; Boza-Serrano, Antonio; Dunning, Christopher J.R.

2018-01-01

Background: Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication...

Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

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Louise C. Laurent

2015-08-01

Full Text Available Extracellular RNAs (exRNAs have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity.

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Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T

2011-04-15

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.

Lindersin B from Lindernia crustacea induces neuritogenesis by activation of tyrosine kinase A/phosphatidylinositol 3 kinase/extracellular signal-regulated kinase signaling pathway.

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Cheng, Lihong; Ye, Ying; Xiang, Lan; Osada, Hiroyuki; Qi, Jianhua

2017-01-15

Neurotrophic factors such as nerve growth factor (NGF) play important roles in nervous system. NGF is a potential therapeutic drug for treatment of neurodegenerative diseases. However, because of physicochemical property, NGF cannot pass through the blood-brain barrier (BBB). Hence, small molecules which exhibit NGF-mimic activity and can pass through the BBB are considered to be promising drug candidates for treatment of such diseases. The present study was designed to isolate NGF-mimic substance from extract of natural products, determine their structures and investigate mechanism of action of the active substance. Extract of Lindernia crustacean was partitioned between water and ethyl acetate to obtain water layer and ethyl acetate layer samples, respectively, and then evaluated their neuritogenic activity in PC12 cells. The active sample was separated by open columns, followed by HPLC purification to obtain active compound. Then, specific inhibitors were used to investigate signaling pathway of neurite outgrowth induced by the active compound. Finally, western blot analysis was performed to confirm the pathway proposed by inhibitor experiments. The ethyl acetate layer sample of extract of Lindernia crustacea exhibited significant neuritogenic activity. Two new compounds, named as linderside A and lindersin B, were isolated; their structures were elucidated by spectroscopic and chemical derivatization methods. Linderside A is a cucurbitane glycoside, whereas lindersin B is a cucurbitane triterpenoid. Each compound has an unusual isopentene unit, namely, a double bond bound to an unmodified isopropyl group at the end of cucurbitane triterpenoid side chain. Among them, lindersin B induced significant neurite outgrowth in PC12 cells, while linderside A was inactive against PC12 cells. Western blotting analysis results showed that lindersin B-induced neuritogenic activity depended on the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal

Lack of direct evidence for a functional role of voltage-operated calcium channels in juxtaglomerular cells

DEFF Research Database (Denmark)

Kurtz, A; Skott, O; Chegini, S

1990-01-01

in patch-clamped nor in intact Furaester-loaded cells. Moreover, basal renin secretion from a preparation enriched in mouse juxtaglomerular cells and from rat glomeruli with attached juxtaglomerular cells was not inhibited when extracellular potassium was isoosmotically increased to 56 mmol/l. In mouse...... kidney slices, however, depolarizing potassium concentrations caused a delayed inhibition at 56 mmol/l and a delayed stimulation of renin secretion at 110 mmol/l. Taken together, our study does not provide direct evidence for a role of voltage-activated calcium channels in the regulation of calcium...

Extracellular Vesicles in Hematological Disorders

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Anat Aharon

2014-10-01

Full Text Available Extracellular vesicles (EVs, comprised of exosomes, microparticles, apoptotic bodies, and other microvesicles, are shed from a variety of cells upon cell activation or apoptosis. EVs promote clot formation, mediate pro-inflammatory processes, transfer proteins and miRNA to cells, and induce cell signaling that regulates cell differentiation, proliferation, migration, invasion, and apoptosis. This paper will review the contribution of EVs in hematological disorders, including hemoglobinopathies (sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, and hematological malignancies (lymphomas, myelomas, and acute and chronic leukemias.

Finite element model to study calcium distribution in oocytes ...

African Journals Online (AJOL)

Calcium is one of the most important signalling ions in cell biology performing numerous functions with high specificity. A calcium wave triggers life at fertilization but also can cause cell death. The means by which this single ion can be both highly specific and universal is believed to lie in its spatiotemporal dynamics ...

Effects of Human Mesenchymal Stem Cells Coculture on Calcium-Induced Differentiation of Normal Human Keratinocytes.

Science.gov (United States)

Sah, Shyam Kishor; Kim, Hae Young; Lee, Ji Hae; Lee, Seong-Wook; Kim, Hyung-Sik; Kim, Yeon-Soo; Kang, Kyung-Sun; Kim, Tae-Yoon

2017-06-01

The influence of mesenchymal stem cells (MSCs) on keratinocytes in altered microenvironments is poorly understood. Here, we cocultured umbilical cord blood-derived MSCs with normal human epidermal keratinocytes to evaluate their paracrine effect in the presence of high extracellular calcium (Ca 2+ ) concentration. High Ca 2+ environment to keratinocytes can disrupt normal skin barrier function due to abnormal/premature differentiation of keratinocytes. Surprisingly, we found that MSCs suppress both proliferation and differentiation of keratinocytes under a high Ca 2+ environment in transforming growth factors β1 (TGFβ1)-dependent manner. Furthermore, we determined that MSCs can regulate the mitogen-activated protein kinases, phosphatidylinositol 3-kinase/protein kinase B, and protein kinase C pathways in Ca 2+ -induced differentiated keratinocytes. Knockdown of TGFβ1 from MSCs results in decreased suppression of differentiation with significantly increased proliferation of keratinocytes compared with control MSCs. MSCs-derived TGFβ1 further induced growth inhibition of keratinocyte in high extracellular Ca 2+ environment as analyzed by a decrease in DNA synthesis, accumulation of phosphorylated retinoblastoma protein, cdc2, and increased mRNA level of p21, and independent of TGFβ1/SMAD pathway. Taken together, we found that MSCs-derived TGFβ1 is a critical regulator of keratinocyte function, and involves multiple proximal signaling cascades. Stem Cells 2017;35:1592-1602. © 2017 AlphaMed Press.

TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

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Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

2013-11-01

The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John

Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay.

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Kopljar, Ivan; Hermans, An N; Teisman, Ard; Gallacher, David J; Lu, Hua Rong

Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model. We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/μCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform. We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline). In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool. Copyright © 2018 Elsevier Inc. All rights reserved.

Lamotrigine blocks NMDA receptor-initiated arachidonic acid signalling in rat brain: Implications for its efficacy in bipolar disorder

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Ramadan, Epolia; Basselin, Mireille; Rao, Jagadeesh S.; Chang, Lisa; Chen, Mei; Ma, Kaizong; Rapoport, Stanley I.

2011-01-01

An upregulated brain arachidonic acid (AA) cascade and a hyperglutamatergic state characterize bipolar disorder (BD). Lamotrigine (LTG), a mood stabilizer approved for treating BD, is reported to interfere with glutamatergic neurotransmission involving N-methyl-D-aspartate receptors (NMDARs). NMDARs allow extracellular calcium into the cell, thereby stimulating calcium-dependent cytosolic phospholipase A2 (cPLA2) to release arachidonic acid (AA) from membrane phospholipid. We hypothesized that LTG, like other approved mood stabilizers, would reduce NMDAR-mediated AA signaling in rat brain. An acute subconvulsant dose of NMDA (25 mg/kg) or saline was administered intraperitoneally to unanesthetized rats that had been treated p.o. daily for 42 days with vehicle or a therapeutically relevant dose of LTG (10 mg/kg/.d). Regional brain AA incorporation coefficients k* and rates Jin, AA signals, were measured using quantitative autoradiography after intravenous [1-14C]AA infusion, as were other AA cascade markers. In chronic vehicle-treated rats, acute NMDA compared to saline increased k* and Jin in widespread regions of the brain, as well as prostaglandin (PG)E2 and thromboxane B2 concentrations. Chronic LTG treatment compared to vehicle reduced brain cyclooxygenase (COX) activity, PGE2 concentration, and DNA binding activity of the COX-2 transcription factor, NF-κB. Pretreatment with chronic LTG blocked the acute NMDA effects on AA cascade markers. In summary, chronic LTG like other mood stabilizers blocks NMDA-mediated signaling involving the AA metabolic cascade. Since markers of the AA cascade and of NMDAR signaling are up-regulated in the postmortem BD brain, mood stabilizers generally may be effective in BD by dampening NMDAR signalling and the AA cascade. PMID:21733229

Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

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Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

2008-04-01

A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH 2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH 2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

International Nuclear Information System (INIS)

Isobe, Ichiro; Maeno, Yoshitaka; Nagao, Masataka; Iwasa, Mineo; Koyama, Hiroyoshi; Seko-Nakamura, Yoshimi; Monma-Ohtaki, Jun

2003-01-01

There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

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Mazars, C; Thion, L; Thuleau, P; Graziana, A; Knight, M R; Moreau, M; Ranjeva, R

1997-11-01

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

A signal processing analysis of Purkinje cells in vitro

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Ze'ev R Abrams

2010-05-01

Full Text Available Cerebellar Purkinje cells in vitro fire recurrent sequences of Sodium and Calcium spikes. Here, we analyze the Purkinje cell using harmonic analysis, and our experiments reveal that its output signal is comprised of three distinct frequency bands, which are combined using Amplitude and Frequency Modulation (AM/FM. We find that the three characteristic frequencies - Sodium, Calcium and Switching – occur in various combinations in all waveforms observed using whole-cell current clamp recordings. We found that the Calcium frequency can display a frequency doubling of its frequency mode, and the Switching frequency can act as a possible generator of pauses that are typically seen in Purkinje output recordings. Using a reversibly photo-switchable kainate receptor agonist, we demonstrate the external modulation of the Calcium and Switching frequencies. These experiments and Fourier analysis suggest that the Purkinje cell can be understood as a harmonic signal oscillator, enabling a higher level of interpretation of Purkinje signaling based on modern signal processing techniques.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

Energy Technology Data Exchange (ETDEWEB)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

International Nuclear Information System (INIS)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

2015-01-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

DESENSITIZATION PROPERTIES OF P2X3 RECEPTORS SHAPING PAIN SIGNALLING

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Rashid eGiniatullin

2013-12-01

Full Text Available ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called ‘high affinity desensitization’. We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called ‘left flipper’ converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given such their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain.

In vivo immunotoxicity of perfluorooctane sulfonate in BALB/c mice: Identification of T-cell receptor and calcium-mediated signaling pathway disruption through gene expression profiling of the spleen.

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Lv, Qi-Yan; Wan, Bin; Guo, Liang-Hong; Yang, Yu; Ren, Xiao-Min; Zhang, Hui

2015-10-05

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that is used worldwide and is continuously being detected in biota and the environment, thus presenting potential threats to the ecosystem and human health. Although PFOS is highly immunotoxic, its underlying molecular mechanisms remain largely unknown. The present study examined PFOS-induced immunotoxicity in the mouse spleen and explored its underlying mechanisms by gene expression profiling. Oral exposure of male BALB/c mice for three weeks followed by one-week recovery showed that a 10 mg/kg/day PFOS exposure damaged the splenic architecture, inhibited T-cell proliferation in response to mitogen, and increased the percentages of T helper (CD3(+)CD4(+)) and cytotoxic T (CD3(+)CD8(+)) cells, despite the decrease in the absolute number of these cells. A delayed type of PFOS immunotoxicity was observed, which mainly occurred during the recovery period. Global gene expression profiling of mouse spleens and QRT-PCR analyses suggest that PFOS inhibited the expression of genes involved in cell cycle regulation and NRF2-mediated oxidative stress response, and upregulated those in TCR signaling, calcium signaling, and p38/MAPK signaling pathways. Western blot analysis confirmed that the expressions of CAMK4, THEMIS, and CD3G, which were involved in the upregulated pathways, were induced upon PFOS exposure. Acute PFOS exposure modulated calcium homoeostasis in splenocytes. These results indicate that PFOS exposure can activate TCR signaling and calcium ion influx, which provides a clue for the potential mechanism of PFOS immunotoxicity. The altered signaling pathways by PFOS treatment as revealed in the present study might facilitate in better understanding PFOS immunotoxicity and explain the association between immune disease and PFOS exposure. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

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Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

2006-10-03

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

Differences in signal activation by LH and hCG are mediated by the LH/CG receptor`s extracellular hinge region

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Paul eGrzesik

2015-09-01

Full Text Available The human lutropin/choriogonadotropin receptor (LHCGR can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG - secreted by the placenta, and lutropin (LH - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich repeat domain (LRRD, as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting mutations. These helix preserving modifications showed no effect on hormone induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10 deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region s. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region