Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores

PubMed Central

Sobolewski, Peter; Kandel, Judith; Klinger, Alexandra L.

2011-01-01

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50–150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway. PMID:21633077

The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

PubMed Central

Nolz, Jeffrey C.; Gomez, Timothy S.; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B.; Shimizu, Yoji; Burkhardt, Janis K.; Freedman, Bruce D.; Billadeau, Daniel D.

2007-01-01

Summary Background The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. Results By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and β-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCγ1 activation and IP3-mediated store release. Conclusions These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation. PMID:16401421

The PLC/IP3R/PKC Pathway is Required for Ethanol-enhanced GABA Release

PubMed Central

Kelm, M. Katherine; Weinberg, Richard J.; Criswell, Hugh E.; Breese, George R.

2010-01-01

Summary Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP3Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP3Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP3Rs requires binding of IP3, generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP3R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP3R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells. PMID:20206640

Disruption of the vacuolar calcium-ATPases in arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway

USDA-ARS?s Scientific Manuscript database

Calcium (Ca2+) signals regulate many aspects of plant development, including the Hypersensitive Response (HR) that triggers a programmed cell death response to protect a plant from a pathogen. A transient increase in cytosolic Ca2+ ([Ca2+]cyt ) results from Ca2+ entry from the apoplast or release fr...

Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells

PubMed Central

Balghi, Haouaria; Sebille, Stéphane; Mondin, Ludivine; Cantereau, Anne; Constantin, Bruno; Raymond, Guy; Cognard, Christian

2006-01-01

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling. PMID:16847098

Endomorphin-2 is Released from Newborn Rat Primary Sensory Neurons in a Frequency- and Calcium- Dependent Manner

PubMed Central

Scanlin, Heather L.; Carroll, Elizabeth A.; Jenkins, Victoria K.; Balkowiec, Agnieszka

2008-01-01

Recent evidence indicates that endomorphins, endogenous mu-opioid receptor (MOR) agonists, modulate synaptic transmission in both somatic and visceral sensory pathways. Here we show that endomorphin-2 (END-2) is expressed in newborn rat dorsal root ganglion (DRG) and nodose-petrosal ganglion complex (NPG) neurons, and rarely co-localizes with brain-derived neurotrophic factor (BDNF). In order to examine activity-dependent release of END-2 from neurons, we established a model using dispersed cultures of DRG and NPG cells activated by patterned electrical field stimulation. To detect release of END-2, we developed a novel rapid capture ELISA, in which END-2 capture antibody was added to neuronal cultures shortly before their electrical stimulation. The conventional assay was effective at reliably detecting END-2 only when the cells were stimulated in the presence of CTAP, a MOR-selective antagonist. This suggests that the strength of the novel assay is related primarily to rapid capture of released END-2 before it binds to endogenous MORs. Using the rapid capture ELISA, we found that stimulation protocols known to induce plastic changes at sensory synapses were highly effective at releasing END-2. Removal of extracellular calcium or blocking voltage-activated calcium channels significantly reduced the release. Together, our data provide the first evidence that END-2 is expressed by newborn DRG neurons of all sizes found in this age group, and can be released from these, as well as from NPG neurons, in an activity-dependent manner. These results point to END-2 as a likely mediator of activity-dependent plasticity in sensory pathways. PMID:18513316

Expressed ryanodine receptor can substitute for the inositol 1,4,5-trisphosphate receptor in Xenopus laevis oocytes during progesterone-induced maturation.

PubMed

Kobrinsky, E; Ondrias, K; Marks, A R

1995-12-01

Two structurally related forms of intracellular calcium release channels that can mediate the release of intracellular calcium have been identified: the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate receptors (IP3R). Each channel responds to distinct pathways for activation. The IP3R is activated by IP3 and the RyR is thought to be activated by calcium or by another second messenger cADP ribose. It has been proposed that each type of channel subserves a specialized pool of intracellular calcium, and it is not understood why some cell types require more than one form of intracellular calcium release channel. The present study was designed to examine whether the RyR can substitute for the IP3R during oocyte maturation. IP3R expression was inhibited in Xenopus laevis oocytes using antisense oligonucleotides. These oocytes, with reduced levels of IP3R, demonstrated a marked delay in the time course of progesterone-induced maturation. The cloned skeletal muscle RyR1 was then expressed in X. laevis oocytes that were deficient in IP3R. Functional studies showed that the properties of the cloned RyR1, expressed in oocytes, were comparable to those of the native RyR1. X. laevis oocytes deficient in IP3R, but expressing RyR1, were able to undergo progesterone-induced maturation with a time course comparable to that seen in wild-type oocytes when caffeine was used to activate RyR and induce intracellular calcium release. These studies show that RyR1 can substitute for the IP3R as the intracellular calcium release channel required for Xenopus oocyte maturation and that intracellular calcium release is important for controlling the rate of progesterone-induced maturation.

Regulation of ACh release from guinea pig bladder urothelial cells: potential role in bladder filling sensations.

PubMed

McLatchie, L M; Young, J S; Fry, C H

2014-07-01

The aim of this study was to quantify and characterize the mechanism of non-neuronal ACh release from bladder urothelial cells and to determine if urothelial cells could be a site of action of anti-muscarinic drugs. A novel technique was developed whereby ACh could be measured from freshly isolated guinea pig urothelial cells in suspension following mechanical stimulation. Various agents were used to manipulate possible ACh release pathways in turn and to study the effects of muscarinic receptor activation and inhibition on urothelial ATP release. Minimal mechanical stimulus achieved full ACh release, indicating a small dynamic range and possible all-or-none signal. ACh release involved a mechanism dependent on the anion channel CFTR and intracellular calcium concentration, but was independent of extracellular calcium, vesicular trafficking, connexins or pannexins, organic cation transporters and was not affected by botulinum-A toxin. Stimulating ACh receptors increased ATP production and antagonizing them reduced ATP release, suggesting a link between ACh and ATP release. These results suggest that release of non-neuronal ACh from the urothelium is large enough and well located to act as a modulator of ATP release. It is hypothesized that this pathway may contribute to the actions of anti-muscarinic drugs in reducing the symptoms of lower urinary tract syndromes. Additionally the involvement of CFTR in ACh release suggests an exciting new direction for the treatment of these conditions. © 2014 The British Pharmacological Society.

ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle*

PubMed Central

Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

2009-01-01

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. PMID:19822518

The effects of monobromobimane on calcium and phenylarsineoxide-induced mitochondrial swelling and cytochrome C release in isolated brain mitochondria.

PubMed

Abe, Tsutomu; Takagi, Norio; Nakano, Midori; Tanonaka, Kouichi; Takeo, Satoshi

2004-04-01

A possible involvement of inhibitory effects of monobromobimane (MBM), a thiol reagent, on the swelling and the release of cytochrome c in the isolated brain mitochondria was examined. MBM dose-dependently inhibited the calcium and phenylarsineoxide-induced mitochondrial swelling and cytochrome c release. Significant relationships between mitochondrial swelling and cytochrome c release were detected. Furthermore, effects of in vivo treatment with MBM on neuronal cell damage after transient (15 min) global ischemia in rats were examined. Infusion of MBM (1 or 3 microg/animal) to cerebral ventricles attenuated an increased number of TUNEL-positive cells and neuronal cell death in the hippocampal CA1 region at 72 h of reperfusion. These results suggest that MBM may have an ability to inhibit mitochondria-associated apoptotic pathways through attenuation of the mitochondrial swelling and the release of cytochrome c.

Focal adhesion kinase dependent activation of the PI3 kinase pathway by the functional soluble form of neurotensin receptor-3 in HT29 cells.

PubMed

Massa, Fabienne; Devader, Christelle; Béraud-Dufour, Sophie; Brau, Frédéric; Coppola, Thierry; Mazella, Jean

2013-05-01

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin, is thought to display several functions including a role as a receptor or a co-receptor, in the sorting to plasma membrane and to lysosomes, and in the regulated secretion. The aim of this study was to investigate the function of the soluble form of NTSR3 (sNTSR3) released from several cell lines including colonic cancer cells. The human adenocarcinoma epithelial cell line HT29 has been used to monitor the release, the binding and internalization of sNTSR3 by radioreceptor assays and confocal microscopy. The modulation of the intracellular signaling pathways by the protein has been investigated by using Fura-2 fluorescence calcium imaging microscopy and Western blots analysis. We demonstrated that sNTSR3 specifically binds and internalizes into HT29 cells. This binding, independent from the transactivation of the epidermal growth factor receptor, leads to the increase of intracellular calcium concentration and to the activation of a FAK/Src-dependent activation of the PI3 kinase pathway. In conclusion, sNTSR3 released from the membrane bound NTSR3 is a functional protein able to activate intracellular pathways involved in cell survival but probably not in cell growth. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Traffic-related PM2.5 regulates IL-2 releasing in Jurkat T cells by calcium signaling pathway].

PubMed

Tong, Guoqiang; Zhang, Zhihong; Han, Jianbiao; Qiu, Yong; Xu, Jianjun

2013-09-01

To explore the effects of traffic-related PM2.5 on interleukin-2 (IL-2) in Jurkat T cells and the regulatory action of calcium signaling pathway. The cells were exposed to 100 microg/ml of PM2.5 for 3, 6 and 24 h. Normal saline group, blank filter group, calcium chelating agent EGTA group and the calcineurin antagonist cyclosporine A (CSA) group were as parallel control. The level of IL-2 was detected by ELISA kits, the mRNA expression of CaN, NFAT were determined by QRT-PCR. The nuclear distribution of NFAT was observed by immunofluorescence microscopy. The level of IL-2 in Jurkat T cells exposed to 100 microg/ml PM2.5 was significantly lower than parallel groups, but higher than PM2.5 + CSA group and PM2.5 + EGTA group (P < 0.05). With the increase of time, the releasing level of IL-2 appeared reducing trend in 100 microg/ml of PM2.5 group. The mRNA expression level of NFAT and CaN were higher than parallel groups, PM2.5 + CSA group and PM2.5 + EGTA group (P < 0.05). PM2.5 can induce NFAT protein with dephosphorylation and be activated, and NFAT protein can shift into nuclear. The level of IL-2 was negatively associated with the expression level of NFAT and CaN gene (P < 0.05). Traffic-related PM2.5 may inhibit the releasing of IL-2, Ca(2+)-CaN-NFAT signal pathway may involve in the regulation of IL-2.

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1.

PubMed

Heidarsson, Pétur O; Naqvi, Mohsin M; Otazo, Mariela R; Mossa, Alessandro; Kragelund, Birthe B; Cecconi, Ciro

2014-09-09

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.

Zinc release from Schaffer collaterals and its significance.

PubMed

Takeda, Atsushi; Nakajima, Satoko; Fuke, Sayuri; Sakurada, Naomi; Minami, Akira; Oku, Naoto

2006-02-15

On the basis of the evidence that approximately 45% of Schaffer collateral boutons are zinc-positive, zinc release from Schaffer collaterals and its action were examined in hippocampal slices. When zinc release from Schaffer collaterals was examined using ZnAF-2, a membrane-impermeable zinc indicator, ZnAF-2 signal in the stratum radiatum of the CA1 was increased by tetanic stimuli at 100 Hz for 1s, suggesting that zinc is released from Schaffer collaterals in a calcium- and impulse-dependent manner. An in vivo microdialysis experiment indicated that the perfusion with 10 microM zinc significantly decreases extracellular glutamate concentration in the CA1. When tetanic stimuli at 100 Hz for 5s were delivered to the dentate granule cells, the increase in calcium signal in the stratum radiatum of the CA1, as well as in the stratum lucidum of the CA3, was attenuated by addition of 10 microM zinc, while enhanced by addition of 1mM CaEDTA, a membrane-impermeable zinc chelator. The increase in calcium signal in the CA1, in which Schaffer collateral synapses exist, during delivery of tetanic stimuli at 100 Hz for 1s to the Schaffer collateral-commissural pathway was also significantly enhanced by addition of 1mM CaEDTA. These results suggest that zinc released from Schaffer collaterals suppressively modulates presynaptic and postsynaptic calcium signaling in the CA1, followed by the suppression of glutamate release.

Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum

NASA Astrophysics Data System (ADS)

Bezprozvanny, Llya; Watras, James; Ehrlich, Barbara E.

1991-06-01

RELEASE of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel1-3 and a calcium-gated channel (ryanodine receptor)4-6. Using specific antibodies, both receptors were found in Purkinje cells of cerebellum7,8. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 µM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 µM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feed-back and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.

Mitochondrial membrane permeabilization and cell death during myocardial infarction: roles of calcium and reactive oxygen species

PubMed Central

Webster, Keith A

2013-01-01

Excess generation of reactive oxygen species (ROS) and cytosolic calcium accumulation play major roles in the initiation of programmed cell death during acute myocardial infarction. Cell death may include necrosis, apoptosis and autophagy, and combinations thereof. During ischemia, calcium handling between the sarcoplasmic reticulum and myofilament is disrupted and calcium is diverted to the mitochondria causing swelling. Reperfusion, while essential for survival, reactivates energy transduction and contractility and causes the release of ROS and additional ionic imbalance. During acute ischemia–reperfusion, the principal death pathways are programmed necrosis and apoptosis through the intrinsic pathway, initiated by the opening of the mitochondrial permeability transition pore and outer mitochondrial membrane permeabilization, respectively. Despite intense investigation, the mechanisms of action and modes of regulation of mitochondrial membrane permeabilization are incompletely understood. Extrinsic apoptosis, necroptosis and autophagy may also contribute to ischemia–reperfusion injury. In this review, the roles of dysregulated calcium and ROS and the contributions of Bcl-2 proteins, as well as mitochondrial morphology in promoting mitochondrial membrane permeability change and the ensuing cell death during myocardial infarction are discussed. PMID:23176689

Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts.

PubMed

Martínez, Javier; Moreno, Juan J

2005-09-01

Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

Purinergic signalling in the enteric nervous system (An overview of current perspectives).

PubMed

King, Brian F

2015-09-01

Purinergic Signalling in the Enteric Nervous System involves the regulated release of ATP (or a structurally-related nucleotide) which activates an extensive suite of membrane-inserted receptors (P2X and P2Y subtypes) on a variety of cell types in the gastrointestinal tract. P2X receptors are gated ion-channels permeable to sodium, potassium and calcium. They depolarise cells, act as a pathway for calcium influx to activate calcium-dependent processes and initiate gene transcription, interact at a molecular level as a form of self-regulation with lipids within the cell wall (e.g. PIP2) and cross-react with other membrane-inserted receptors to regulate their activity (e.g. nAChRs). P2Y receptors are metabotropic receptors that couple to G-proteins. They may release calcium ions from intracellular stores to activate calcium-dependent processes, but also may activate calcium-independent signalling pathways and influence gene transcription. Originally ATP was a candidate only for NANC neurotransmission, for inhibitory motoneurons supplying the muscularis externa of the gastrointestinal tract and bringing about the fast IJP. Purinergic signalling later included neuron-neuron signalling in the ENS, via the production of either fast or slow EPSPs. Later still, purinergic signalling included the neuro-epithelial synapse-for efferent signalling to epithelia cells participating in secretion and absorption, and afferent signalling for chemoreception and mechanoreception at the surface of the mucosa. Many aspects of purinergic signalling have since been addressed in a series of highly-focussed and authoritative reviews. In this overview however, the current focus is on key aspects of purinergic signalling where there remains uncertainty and ambiguity, with the view to stimulating further research in these areas. Copyright © 2015 Elsevier B.V. All rights reserved.

Lipid body accumulation alters calcium signaling dynamics in immune cells

PubMed Central

Greineisen, William E.; Speck, Mark; Shimoda, Lori M.N.; Sung, Carl; Phan, Nolwenn; Maaetoft-Udsen, Kristina; Stokes, Alexander J.; Turner, Helen

2014-01-01

Summary There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcεRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signalling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcεRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signalling pathway and its downstream targets. PMID:25016314

Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

PubMed

Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

2015-12-01

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

Carbon nanopipettes characterize calcium release pathways in breast cancer cells

NASA Astrophysics Data System (ADS)

Schrlau, Michael G.; Brailoiu, Eugen; Patel, Sandip; Gogotsi, Yury; Dun, Nae J.; Bau, Haim H.

2008-08-01

Carbon-based nanoprobes are attractive for minimally invasive cell interrogation but their application in cell physiology has thus far been limited. We have developed carbon nanopipettes (CNPs) with nanoscopic tips and used them to inject calcium-mobilizing messengers into cells without compromising cell viability. We identify pathways sensitive to cyclic adenosine diphosphate ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) in breast carcinoma cells. Our findings demonstrate the superior utility of CNPs for intracellular delivery of impermeant molecules and, more generally, for cell physiology studies. The CNPs do not appear to cause any lasting damage to cells. Their advantages over commonly used glass pipettes include smaller size, breakage and clogging resistance, and potential for multifunctionality such as in concurrent injection and electrical measurements.

Carbon nanopipettes characterize calcium release pathways in breast cancer cells.

PubMed

Schrlau, Michael G; Brailoiu, Eugen; Patel, Sandip; Gogotsi, Yury; Dun, Nae J; Bau, Haim H

2008-08-13

Carbon-based nanoprobes are attractive for minimally invasive cell interrogation but their application in cell physiology has thus far been limited. We have developed carbon nanopipettes (CNPs) with nanoscopic tips and used them to inject calcium-mobilizing messengers into cells without compromising cell viability. We identify pathways sensitive to cyclic adenosine diphosphate ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) in breast carcinoma cells. Our findings demonstrate the superior utility of CNPs for intracellular delivery of impermeant molecules and, more generally, for cell physiology studies. The CNPs do not appear to cause any lasting damage to cells. Their advantages over commonly used glass pipettes include smaller size, breakage and clogging resistance, and potential for multifunctionality such as in concurrent injection and electrical measurements.

Herpes simplex virus triggers activation of calcium-signaling pathways

PubMed Central

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

Influence of the autonomic nervous system on calcium homeostasis in the rat.

PubMed

Stern, J E; Cardinali, D P

1994-01-01

The local surgical manipulation of sympathetic and parasympathetic nerves innervating the thyroid-parathyroid territory was employed to search for the existence of a peripheral neuroendocrine link controlling parathyroid hormone (PTH) and calcitonin (CT) release. From 8 to 24 h after superior cervical ganglionectomy (SCGx), at the time of wallerian degeneration of thyroid-parathyroid sympathetic nerve terminals, an alpha-adrenergic inhibition, together with a minor beta-adrenergic stimulation, of hypercalcemia-induced CT release, and an alpha-adrenoceptor inhibition of hypocalcemia-induced PTH release were found. In chronically SCGx rats PTH response to EDTA was slower, and after CaCl2 injection, serum calcium attained higher levels in face of normal CT levels. SCGx blocked the PTH increase found in sham-operated rats stressed by a subcutaneous injection of turpentine oil, but did not affect the greater response to EDTA. The higher hypocalcemia seen after turpentine oil was no longer observed in SCGx rats. The effects of turpentine oil stress on calcium and CT responses to a bolus injection of CaCl2 persisted in rats subjected to SCGx 14 days earlier. Interruption of thyroid-parathyroid parasympathetic input conveyed by the thyroid nerves (TN) and the inferior laryngeal nerves (ILN) caused a fall in total serum calcium, an increase of PTH levels and a decrease of CT levels, when measured 10 days after surgery. Greater responses of serum CT and PTH were detected in TN-sectioned, and in TN- or ILN-sectioned rats, respectively. Physiological concentrations of CT decreased, and those of PTH increased, in vitro cholinergic activity in rat SCG, measured as specific choline uptake, and acetylcholine synthesis and release. The results indicate that cervical autonomic nerves constitute a pathway through which the brain modulates calcium homeostasis.

P2Y1 receptor antagonists mitigate oxygen and glucose deprivation‑induced astrocyte injury.

PubMed

Guo, Hui; Liu, Zhong-Qiang; Zhou, Hui; Wang, Zhi-Ling; Tao, Yu-Hong; Tong, Yu

2018-01-01

The aim of the present study was to elucidate the effects of blocking the calcium signaling pathway of astrocytes (ASs) on oxygen and glucose deprivation (OGD)‑induced AS injury. The association between the changes in the concentrations of AS‑derived transmitter ATP and glutamic acid, and the changes in calcium signaling under the challenge of OGD were investigated. The cortical ASs of Sprague Dawley rats were cultured to establish the OGD models of ASs. The extracellular concentrations of ATP and glutamic acid in the normal group and the OGD group were detected, and the intracellular concentration of calcium ions (Ca2+) was detected. The effects of 2'‑deoxy‑N6‑methyl adenosine 3', 5'‑diphosphate diammonium salt (MRS2179), a P2Y1 receptor antagonist, on the release of calcium and glutamic acid of ASs under the condition of OGD were observed. The OGD challenge induced the release of glutamic acid and ATP by ASs in a time‑dependent manner, whereas elevation in the concentration of glutamic acid lagged behind that of the ATP and Ca2+. The concentration of Ca2+ inside ASs peaked 16 h after OGD, following which the concentration of Ca2+ was decreased. The effects of elevated release of glutamic acid by ASs when challenged by OGD may be blocked by MRS2179, a P2Y1 receptor antagonist. Furthermore, MRS2179 may significantly mitigate OGD‑induced AS injury and increase cell survival. The ASs of rats cultured in vitro expressed P2Y1 receptors, which may inhibit excessive elevation in the concentration of intracellular Ca2+. Avoidance of intracellular calcium overload and the excessive release of glutamic acid may be an important reason why MRS2179 mitigates OGD‑induced AS injury.

Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and independent effects

USDA-ARS?s Scientific Manuscript database

Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress and death pathways in a skeletal muscle model. Diffe...

Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels.

PubMed

Jarvis, Scott E; Zamponi, Gerald W

2005-05-01

Calcium entry through presynaptic voltage-gated calcium channels is essential for neurotransmitter release. The two major types of presynaptic calcium channels contain a synaptic protein interaction site that physically interacts with synaptic vesicle release proteins. This is thought to tighten the coupling between the sources of calcium entry and the neurotransmitter release machinery. Conversely, the binding of synaptic proteins to presynaptic calcium channels regulates calcium channel activity. Hence, presynaptic calcium channels act not only as the masters of the synaptic release process, but also as key targets for feedback inhibition.

Melatonin regulates somatotrope and lactotrope function through common and distinct signaling pathways in cultured primary pituitary cells from female primates.

PubMed

Ibáñez-Costa, Alejandro; Córdoba-Chacón, José; Gahete, Manuel D; Kineman, Rhonda D; Castaño, Justo P; Luque, Raúl M

2015-03-01

Melatonin (MT) is secreted by the pineal gland and exhibits a striking circadian rhythm in its release. Depending on the species studied, some pituitary hormones also display marked circadian/seasonal patterns and rhythms of secretion. However, the precise relationship between MT and pituitary function remains controversial, and studies focusing on the direct role of MT in normal pituitary cells are limited to nonprimate species. Here, adult normal primate (baboons) primary pituitary cell cultures were used to determine the direct impact of MT on the functioning of all pituitary cell types from the pars distalis. MT increased GH and prolactin (PRL) expression/release in a dose- and time-dependent fashion, a response that was blocked by somatostatin. However, MT did not significantly affect ACTH, FSH, LH, or TSH expression/release. MT did not alter GHRH- or ghrelin-induced GH and/or PRL secretions, suggesting that MT may activate similar signaling pathways as ghrelin/GHRH. The effects of MT on GH/PRL release, which are likely mediated through MT1 receptor, involve both common (adenylyl cyclase/protein kinase A/extracellular calcium-channels) and distinct (phospholipase C/intracellular calcium-channels) signaling pathways. Actions of MT on pituitary cells also included regulation of the expression of other key components for the control of somatotrope/lactotrope function (GHRH, ghrelin, and somatostatin receptors). These results show, for the first time in a primate model, that MT directly regulates somatotrope/lactotrope function, thereby lending support to the notion that the actions of MT on these cells might substantially contribute to the define daily patterns of GH and PRL observed in primates and perhaps in humans.

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grondin, Melanie; Marion, Michel; Denizeau, Francine

2007-07-01

Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad frommore » the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl{sup -}/HCO{sub 3} {sup -} exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkes, J.M.; Kajimura, M.; Scott, D.R.

Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide, with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of themore » H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations, suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the (Ca)i transient.« less

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

PubMed

Chang, Mei-Chi; Lin, Szu-I; Lin, Li-Deh; Chan, Chiu-Po; Lee, Ming-Shu; Wang, Tong-Mei; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

2016-04-01

Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Calcium pathway machinery at fertilization in echinoderms

PubMed Central

Ramos, Isabela; Wessel, Gary M.

2016-01-01

Calcium signaling in cells directs diverse physiological processes. The calcium waves triggered by fertilization is a highly conserved calcium signaling event essential for egg activation, and has been documented in every egg tested. This activity is one of the few highly conserved events of egg activation through the course of evolution. Echinoderm eggs, as well as many other cell types, have three main intracellular Ca2+ mobilizing messengers – IP3, cADPR and NAADP. Both cADPR and NAADP were identified as Ca2+ mobilizing messengers using the sea urchin egg homogenate, and this experimental system, along with the intact urchin and starfish oocyte/egg, continues to be a vital tool for investigating the mechanism of action of calcium signals. While many of the major regulatory steps of the IP3 pathway are well resolved, both cADPR and NAADP remain understudied in terms of our understanding of the fundamental process of egg activation at fertilization. Recently, NAADP has been shown to trigger Ca2+ release from acidic vesicles, separately from the ER, and a new class of calcium channels, the two-pore channels (TPCs), was identified as the likely targets for this messenger. Moreover, it was found that both cADPR and NAADP can be synthesized by the same family of enzymes, the ADP-rybosyl cyclases (ARCs). In this context of increasing amount of information, the potential coupling and functional roles of different messengers, intracellular stores and channels in the formation of the fertilization calcium wave in echinoderms will be critically evaluated. PMID:23218671

Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production.

PubMed

Bordji, Karim; Becerril-Ortega, Javier; Nicole, Olivier; Buisson, Alain

2010-11-24

Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

PubMed Central

Helm, Jared R.; Bentley, Marvin; Thorsen, Kevin D.; Wang, Ting; Foltz, Lauren; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C.

2014-01-01

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery. PMID:25006245

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.

PubMed

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.

The oxalate-carbonate pathway: at the interface between biology and geology

NASA Astrophysics Data System (ADS)

Junier, P.; Cailleau, G.; Martin, G.; Guggiari, M.; Bravo, D.; Clerc, M.; Aragno, M.; Job, D.; Verrecchia, E.

2012-04-01

The formation of calcite in otherwise carbonate-free acidic soils through the biological degradation of oxalate is a mechanism termed oxalate-carbonate pathway. This pathway lies at the interface between biological and geological systems and constitutes an important, although underestimated, soil mineral carbon sink. In this case, atmospheric CO2 is fixed by the photosynthetic activity of oxalogenic plants, which is partly destined to the production of oxalate used for the chelation of metals, and particularly, calcium. Fungi are also able to produce oxalate to cope with elevated concentrations of metals. In spite of its abundance as a substrate, oxalate is a very stable organic anion that can be metabolized only by a group of bacteria that use it as carbon and energy sources. These bacteria close the biological cycle by degrading calcium oxalate, releasing Ca2+ and inducing a change in local soil pH. If parameters are favourable, the geological part of the pathway begins, because this change in pH will indirectly lead to the precipitation of secondary calcium carbonate (calcite) in unexpected geological conditions. Due to the initial acidic soil conditions, and the absence of geological carbonate in the basement, it is unexpected to find C in the form of calcite. The activity of the oxalate-carbonate pathway has now been demonstrated in several places around the world, suggesting that its importance can be even greater than expected. In addition, new roles for each of the biological players of the pathway have been revealed recently forcing us to reconsider a global biogeochemical model for oxalate cycling.

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed Central

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-01-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways. PMID:9276720

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-09-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways.

Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato

ERIC Educational Resources Information Center

Liang, Willmann

2008-01-01

This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy (CIPN)

PubMed Central

Canta, Annalisa; Pozzi, Eleonora; Carozzi, Valentina Alda

2015-01-01

The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN). This is due to a related dysregulation of pathways involving calcium signalling, reactive oxygen species and apoptosis. Vincristine is able to affect calcium movement through the Dorsal Root Ganglia (DRG) neuronal mitochondrial membrane, altering its homeostasis and leading to abnormal neuronal excitability. Paclitaxel induces the opening of the mitochondrial permeability transition pore in axons followed by mitochondrial membrane potential loss, increased reactive oxygen species generation, ATP level reduction, calcium release and mitochondrial swelling. Cisplatin and oxaliplatin form adducts with mitochondrial DNA producing inhibition of replication, disruption of transcription and morphological abnormalities within mitochondria in DRG neurons, leading to a gradual energy failure. Bortezomib is able to modify mitochondrial calcium homeostasis and mitochondrial respiratory chain. Moreover, the expression of a certain number of genes, including those controlling mitochondrial functions, was altered in patients with bortezomib-induced peripheral neuropathy. PMID:29056658

Ion release and mechanical properties of calcium silicate and calcium hydroxide materials used for pulp capping.

PubMed

Natale, L C; Rodrigues, M C; Xavier, T A; Simões, A; de Souza, D N; Braga, R R

2015-01-01

To compare the ion release and mechanical properties of a calcium hydroxide (Dycal) and two calcium silicate (MTA Angelus and Biodentine) cements. Calcium and hydroxyl ion release in water from 24-h set cements were calculated from titration with HCl (n = 3). Calcium release after 7, 14, 21 and 28 days at pH 5.5 and 7.0 was measured using ICP-OES (n = 6). Flexural strength (FS) and modulus (E) were tested after 48-h storage, and compressive strength (CS) was tested after 48 h and 7 days (n = 10). Ion release and mechanical data were subjected to anova/Tukey and Kruskal-Wallis/Mann-Whitney tests, respectively (α = 0.05). Titration curves revealed that Dycal released significantly fewer ions in solution than calcium silicates (P < 0.001). Calcium release remained constant at pH 7.0, whilst at pH 5.5, it dropped significantly by 24% after 21 days (P < 0.05). At pH 5.5, MTA Angelus released significantly more calcium than Dycal (P < 0.01), whilst Biodentine had superior ion release than Dycal at pH 7.0 (P < 0.01). Biodentine had superior flexural strength, flexural modulus and compressive strength than the other cements, whilst MTA Angelus had higher modulus than Dycal (P < 0.001). Immediate calcium and hydroxyl ion release in solution was significantly lower for Dycal. In general, all materials released constant calcium levels over 28 days, but release from Dycal was significantly lower than Biodentine and MTA Angelus depending on pH conditions. Biodentine had substantially higher strength and modulus than MTA Angelus and Dycal, both of which demonstrated low stress-bearing capabilities. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Spatial separation of two different pathways accounting for the generation of calcium signals in astrocytes.

PubMed

Oschmann, Franziska; Mergenthaler, Konstantin; Jungnickel, Evelyn; Obermayer, Klaus

2017-02-01

Astrocytes integrate and process synaptic information and exhibit calcium (Ca2+) signals in response to incoming information from neighboring synapses. The generation of Ca2+ signals is mostly attributed to Ca2+ release from internal Ca2+ stores evoked by an elevated metabotropic glutamate receptor (mGluR) activity. Different experimental results associated the generation of Ca2+ signals to the activity of the glutamate transporter (GluT). The GluT itself does not influence the intracellular Ca2+ concentration, but it indirectly activates Ca2+ entry over the membrane. A closer look into Ca2+ signaling in different astrocytic compartments revealed a spatial separation of those two pathways. Ca2+ signals in the soma are mainly generated by Ca2+ release from internal Ca2+ stores (mGluR-dependent pathway). In astrocytic compartments close to the synapse most Ca2+ signals are evoked by Ca2+ entry over the plasma membrane (GluT-dependent pathway). This assumption is supported by the finding, that the volume ratio between the internal Ca2+ store and the intracellular space decreases from the soma towards the synapse. We extended a model for mGluR-dependent Ca2+ signals in astrocytes with the GluT-dependent pathway. Additionally, we included the volume ratio between the internal Ca2+ store and the intracellular compartment into the model in order to analyze Ca2+ signals either in the soma or close to the synapse. Our model results confirm the spatial separation of the mGluR- and GluT-dependent pathways along the astrocytic process. The model allows to study the binary Ca2+ response during a block of either of both pathways. Moreover, the model contributes to a better understanding of the impact of channel densities on the interaction of both pathways and on the Ca2+ signal.

Necroptosis Execution Is Mediated by Plasma Membrane Nanopores Independent of Calcium.

PubMed

Ros, Uris; Peña-Blanco, Aida; Hänggi, Kay; Kunzendorf, Ulrich; Krautwald, Stefan; Wong, W Wei-Lynn; García-Sáez, Ana J

2017-04-04

Necroptosis is a form of regulated necrosis that results in cell death and content release after plasma membrane permeabilization. However, little is known about the molecular events responsible for the disruption of the plasma membrane. Here, we find that early increase in cytosolic calcium in TNF-induced necroptosis is mediated by treatment with a Smac mimetic via the TNF/RIP1/TAK1 survival pathway. This does not require the activation of the necrosome and is dispensable for necroptosis. Necroptosis induced by the activation of TLR3/4 pathways does not trigger early calcium flux. We also demonstrate that necroptotic plasma membrane rupture is mediated by osmotic forces and membrane pores around 4 nm in diameter. This late permeabilization step represents a hallmark in necroptosis execution that is cell and treatment independent and requires the RIP1/RIP3/MLKL core. In support of this, treatment with osmoprotectants reduces cell damage in an in vivo necroptosis model of ischemia-reperfusion injury. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Intracellular sphingosine releases calcium from lysosomes.

PubMed

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.

Thyroid C-Cell Biology and Oncogenic Transformation

PubMed Central

Cote, Gilbert J.; Grubbs, Elizabeth G.; Hofmann, Marie-Claude

2017-01-01

The thyroid parafollicular cell, or commonly named “C-cell,” functions in serum calcium homeostasis. Elevations in serum calcium trigger release of calcitonin from the C-cell, which in turn functions to inhibit absorption of calcium by the intestine, resorption of bone by the osteoclast, and reabsorption of calcium by renal tubular cells. Oncogenic transformation of the thyroid C-cell is thought to progress through a hyperplastic process prior to malignancy with increasing levels of serum calcitonin serving as a biomarker for tumor burden. The discovery that Multiple Endocrine Neoplasia, type 2 is caused by activating mutations of the RET gene serves to highlight the RET-RAS-MAPK signaling pathway in both initiation and progression of medullary thyroid carcinoma. Thyroid C-cells are known to express RET at high levels relative to most cell types, therefore aberrant activation of this receptor is targeted primarily to the C-cell, providing one possible cause of tissue-specific oncogenesis. The role of RET signaling in normal C-cell function is unknown though calcitonin gene transcription appears to be sensitive to RET activation. Beyond RET the modeling of oncogenesis in animals and screening of human tumors for candidate gene mutations has uncovered mutation of RAS family members and inactivation of Rb1 regulatory pathway as potential mediators of C-cell transformation. A growing understanding of how RET interacts with these pathways, both in normal C-cell function and during oncogenic transformation will help in the development of novel molecular targeted therapies. PMID:26494382

Anti-inflammatory effects of secondary metabolites of marine Pseudomonas sp. in human neutrophils are through inhibiting P38 MAPK, JNK, and calcium pathways.

PubMed

Yang, Shun-Chin; Sung, Ping-Jyun; Lin, Chwan-Fwu; Kuo, Jimmy; Chen, Chun-Yu; Hwang, Tsong-Long

2014-01-01

Activated neutrophils play a significant role in the pathogenesis of many inflammatory diseases. The metabolites of marine microorganisms are increasingly employed as sources for developing new drugs; however, very few marine drugs have been studied in human neutrophils. Herein, we showed that secondary metabolites of marine Pseudomonas sp. (N11) significantly inhibited superoxide anion generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils, with IC50 values of 0.67±0.38 µg/ml and 0.84±0.12 µg/ml, respectively. In cell-free systems, neither superoxide anion-scavenging effect nor inhibition of elastase activity was associated with the suppressive effects of N11. N11 inhibited the phosphorylation of p38 MAP kinase and JNK, but not Erk and Akt, in FMLP-induced human neutrophils. Also, N11 dose-dependently attenuated the transient elevation of intracellular calcium concentration in activated neutrophils. In contrast, N11 failed to alter phorbol myristate acetate-induced superoxide anion generation, and the inhibitory effects of N11 were not reversed by protein kinase A inhibitor. In conclusion, the anti-inflammatory effects of N11 on superoxide anion generation and elastase release in activated human neutrophils are through inhibiting p38 MAP kinase, JNK, and calcium pathways. Our results suggest that N11 has the potential to be developed to treat neutrophil-mediated inflammatory diseases.

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

PubMed

Qureshi, Farhan M; Dejene, Eden A; Corbin, Kathryn L; Nunemaker, Craig S

2015-05-01

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Silent synapses in neuromuscular junction development.

PubMed

Tomàs, Josep; Santafé, Manel M; Lanuza, Maria A; García, Neus; Besalduch, Nuria; Tomàs, Marta

2011-01-01

In the last few years, evidence has been found to suggest that some synaptic contacts become silent but can be functionally recruited before they completely retract during postnatal synapse elimination in muscle. The physiological mechanism of developmental synapse elimination may be better understood by studying this synapse recruitment. This Mini-Review collects previously published data and new results to propose a molecular mechanism for axonal disconnection. The mechanism is based on protein kinase C (PKC)-dependent inhibition of acetylcholine (ACh) release. PKC activity may be stimulated by a methoctramine-sensitive M2-type muscarinic receptor and by calcium inflow though P/Q- and L-type voltage-dependent calcium channels. In addition, tropomyosin-related tyrosine kinase B (trkB) receptor-mediated brain-derived neurotrophic factor (BDNF) activity may oppose the PKC-mediated ACh release depression. Thus, a balance between trkB and muscarinic pathways may contribute to the final functional suppression of some neuromuscular synapses during development. © 2010 Wiley-Liss, Inc.

Calsequestrin mediates changes in spontaneous calcium release profiles.

PubMed

Tania, Nessy; Keener, James P

2010-08-07

Calsequestrin (CSQ) is the primary calcium buffer within the sarcoplasmic reticulum (SR) of cardiac cells. It has also been identified as a regulator of Ryanodine receptor (RyR) calcium release channels by serving as a SR luminal sensor. When calsequestrin is free and unbound to calcium, it can bind to RyR and desensitize the channel from cytoplasmic calcium activation. In this paper, we study the role of CSQ as a buffer and RyR luminal sensor using a mechanistic model of RyR-CSQ interaction. By using various asymptotic approximations and mean first exit time calculation, we derive a minimal model of a calcium release unit which includes CSQ dependence. Using this model, we then analyze the effect of changing CSQ expression on the calcium release profile and the rate of spontaneous calcium release. We show that because of its buffering capability, increasing CSQ increases the spark duration and size. However, because of luminal sensing effects, increasing CSQ depresses the basal spark rate and increases the critical SR level for calcium release termination. Finally, we show that with increased bulk cytoplasmic calcium concentration, the CRU model exhibits deterministic oscillations.

Tumor-triggered drug release from calcium carbonate-encapsulated gold nanostars for near-infrared photodynamic/photothermal combination antitumor therapy.

PubMed

Liu, Yanlei; Zhi, Xiao; Yang, Meng; Zhang, Jingpu; Lin, Lingnan; Zhao, Xin; Hou, Wenxiu; Zhang, Chunlei; Zhang, Qian; Pan, Fei; Alfranca, Gabriel; Yang, Yuming; de la Fuente, Jesús M; Ni, Jian; Cui, Daxiang

2017-01-01

Different stimulus including pH, light and temperature have been used for controlled drug release to prevent drug inactivation and minimize side-effects. Herein a novel nano-platform (GNS@CaCO 3 /ICG) consisting of calcium carbonate-encapsulated gold nanostars loaded with ICG was established to couple the photothermal properties of gold nanostars (GNSs) and the photodynamic properties of indocyanine green (ICG) in the photodynamic/photothermal combination therapy (PDT/PTT). In this study, the calcium carbonate worked not only a drug keeper to entrap ICG on the surface of GNSs in the form of a stable aggregate which was protected from blood clearance, but also as the a pH-responder to achieve highly effective tumor-triggered drug release locally. The application of GNS@CaCO 3 /ICG for in vitro and in vivo therapy achieved the combined antitumor effects upon the NIR irradiation, which was superior to the single PDT or PTT. Meanwhile, the distinct pH-triggered drug release performance of GNS@CaCO 3 /ICG implemented the tumor-targeted NIR fluorescence imaging. In addition, we monitored the bio-distribution and excretion pathway of GNS@CaCO 3 /ICG based on the NIR fluorescence from ICG and two-photon fluorescence and photoacoustic signal from GNSs, and the results proved that GNS@CaCO 3 /ICG had a great ability for tumor-specific and tumor-triggered drug release. We therefore conclude that the GNS@CaCO 3 /ICG holds great promise for clinical applications in anti-tumor therapy with tumor imaging or drug tracing.

Selective activation of group III metabotropic glutamate receptor subtypes produces different patterns of γ-aminobutyric acid immunoreactivity and glutamate release in the retina.

PubMed

Guimarães-Souza, E M; Calaza, K C

2012-12-01

Glutamate, the major excitatory neurotransmitter in the retina, functions by activation of both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. Group III mGluRs, except for mGluR6, are mostly found in the inner plexiform layer (IPL), and their retinal functions are not well known. Therefore, we decided to investigate the effect of mGluRIII on glutamate release and GABAergic amacrine cells in the chick retina. The nonselective mGluRIII agonist L-SOP promoted a decrease in the number of γ-aminobutyric acid (GABA)-positive cells and in the GABA immunoreactivity in all sublayers of the IPL. This effect was prevented by the antagonist MAP-4, by GAT-1 inhibitor, and by antagonists of iGluR. Under the conditions used, L-SOP did not alter endogenous glutamate release. VU0155041, an mGluR4-positive allosteric modulator, reduced GABA immunoreactivity in amacrine cells and in sublayers 2 and 4 of the IPL but evoked an increase in the glutamate released. VU0155041's effect was inhibited by the absence of calcium. AMN082, a selective mGluR7-positive allosteric modulator, also decreased GABA immunoreactivity in amacrine cells and sublayers 1, 2, and 3 and increased glutamate release, and this effect was also inhibited by calcium absence. DCPG, an mGluR8-selective agonist, did not significantly alter GABA immunoreactivity in amacrine cells or glutamate release. However, it did significantly increase GABA immunoreactivity in sublayers 4 and 5. The results suggest that mGluRIIIs are involved in the modulation of glutamate and GABA release in the retina, possibly participating in distinct visual pathways: mGluR4 might be involved with cholinergic circuitry, whereas mGluR7 and mGluR8 might participate, respectively, in the OFF and the ON pathways. Copyright © 2012 Wiley Periodicals, Inc.

An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma.

PubMed

Ren, Wei; Beebe, Stephen J

2011-04-01

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55-60%) and calcium-dependent (40-45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway.

PubMed

Willer, Elisabeth A; Malli, Roland; Bondarenko, Alexander I; Zahler, Stefan; Vollmar, Angelika M; Graier, Wolfgang F; Fürst, Robert

2012-10-01

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442. Copyright © 2012 Elsevier Ltd. All rights reserved.

Intracellular sphingosine releases calcium from lysosomes

PubMed Central

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

2015-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

Presynaptic strontium dynamics and synaptic transmission.

PubMed Central

Xu-Friedman, M A; Regehr, W G

1999-01-01

Strontium can replace calcium in triggering neurotransmitter release, although peak release is reduced and the duration of release is prolonged. Strontium has therefore become useful in probing release, but its mechanism of action is not well understood. Here we study the action of strontium at the granule cell to Purkinje cell synapse in mouse cerebellar slices. Presynaptic residual strontium levels were monitored with fluorescent indicators, which all responded to strontium (fura-2, calcium orange, fura-2FF, magnesium green, and mag-fura-5). When calcium was replaced by equimolar concentrations of strontium in the external bath, strontium and calcium both entered presynaptic terminals. Contaminating calcium was eliminated by including EGTA in the extracellular bath, or by loading parallel fibers with EGTA, enabling the actions of strontium to be studied in isolation. After a single stimulus, strontium reached higher peak free levels than did calcium (approximately 1.7 times greater), and decayed more slowly (half-decay time 189 ms for strontium and 32 ms for calcium). These differences in calcium and strontium dynamics are likely a consequence of greater strontium permeability through calcium channels, lower affinity of the endogenous buffer for strontium, and less efficient extrusion of strontium. Measurements of presynaptic divalent levels help to explain properties of release evoked by strontium. Parallel fiber synaptic currents triggered by strontium are smaller in amplitude and longer in duration than those triggered by calcium. In both calcium and strontium, release consists of two components, one more steeply dependent on divalent levels than the other. Strontium drives both components less effectively than does calcium, suggesting that the affinities of the sensors involved in both phases of release are lower for strontium than for calcium. Thus, the larger and slower strontium transients account for the prominent slow component of release triggered by strontium. PMID:10096899

Interactions between calcium precipitation and the polyphosphate-accumulating bacteria metabolism.

PubMed

Barat, R; Montoya, T; Borrás, L; Ferrer, J; Seco, A

2008-07-01

A sequencing batch reactor that is operated for biological phosphorus removal has been operated under different influent calcium concentrations to study the precipitation process and the possible effects of phosphorus precipitation in the biological phosphorus removal process. Four experiments were carried out under different influent calcium concentrations ranging from 10 to 90 g Ca m(-3). The experimental results and the equilibrium study, which are based on the saturation index calculation, confirm that the process controlling the calcium behaviour is the calcium phosphate precipitation. This precipitation takes place at two stages: initially, precipitation of the amorphous calcium phosphate, and later crystallization of hydroxyapatite. Also the accumulation of phosphorus precipitated was observed when the influent calcium concentration was increased. In all the experiments, the influent wastewater ratio P/COD was kept constant. It has been observed that, at high calcium concentration, the ratio between phosphate release and acetate uptake (P(rel)/Ac(uptake)) decreases. Changes in the polyphosphate-accumulating organism (PAO) population and in the glycogen-accumulating organism (GAO) population during the experimental period were ruled out by means of fluorescence in situ hybridization. These results could suggest that PAO are able to change their metabolic pathways based on external conditions, such as influent calcium concentration. The accumulation of phosphorus precipitated as calcium phosphate at high influent calcium concentration throughout the experimental period confirmed that phosphate precipitation is a process that can affect the PAO metabolism.

Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation.

PubMed

Huang, Hsueh-Meei; Zhang, Hui; Xu, Hui; Gibson, Gary E

2003-01-20

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.

The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

PubMed

Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

2016-02-01

In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental Cements.

PubMed

Rajasekharan, Sivaprakash; Vercruysse, Chris; Martens, Luc; Verbeeck, Ronald

2018-01-13

Tricalcium silicate cements (TSC) are used in dental traumatology and endodontics for their bioactivity which is mostly attributed to formation of calcium hydroxide during TSC hydration and its subsequent release of calcium and hydroxide ions. The aim of this study was to determine the effect of volume (Vol), exposed surface area (ESA) and pH of surrounding medium on calcium ion release. Three commercially available hydraulic alkaline dental cements were mixed and condensed into cylindrical tubes of varying length and diameter ( n = 6/group). For the effect of ESA and Vol, tubes were immersed in 10 mL of deionized water. To analyze the effect of environmental pH, the tubes were randomly immersed in 10 mL of buffer solutions with varying pH (10.4, 7.4 or 4.4). The solutions were collected and renewed at various time intervals. pH and/or calcium ion release was measured using a pH glass electrode and atomic absorption spectrophotometer respectively. The change of pH, short-term calcium ion release and rate at which calcium ion release reaches maximum were dependent on ESA ( p < 0.05) while maximum calcium ion release was dependent on Vol of TSC ( p < 0.05). Maximum calcium ion release was significantly higher in acidic solution followed by neutral and alkaline solution ( p < 0.05).

Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental Cements

PubMed Central

Rajasekharan, Sivaprakash; Vercruysse, Chris; Martens, Luc; Verbeeck, Ronald

2018-01-01

Tricalcium silicate cements (TSC) are used in dental traumatology and endodontics for their bioactivity which is mostly attributed to formation of calcium hydroxide during TSC hydration and its subsequent release of calcium and hydroxide ions. The aim of this study was to determine the effect of volume (Vol), exposed surface area (ESA) and pH of surrounding medium on calcium ion release. Three commercially available hydraulic alkaline dental cements were mixed and condensed into cylindrical tubes of varying length and diameter (n = 6/group). For the effect of ESA and Vol, tubes were immersed in 10 mL of deionized water. To analyze the effect of environmental pH, the tubes were randomly immersed in 10 mL of buffer solutions with varying pH (10.4, 7.4 or 4.4). The solutions were collected and renewed at various time intervals. pH and/or calcium ion release was measured using a pH glass electrode and atomic absorption spectrophotometer respectively. The change of pH, short-term calcium ion release and rate at which calcium ion release reaches maximum were dependent on ESA (p < 0.05) while maximum calcium ion release was dependent on Vol of TSC (p < 0.05). Maximum calcium ion release was significantly higher in acidic solution followed by neutral and alkaline solution (p < 0.05). PMID:29342837

Glutamate and Dynorphin Release from a Subcellular Fraction Enriched in Hippocampal Mossy Fiber Synaptosomes

DTIC Science & Technology

1988-01-01

presence of extrasynaptosomal calcium . while only 3(0- of the evoked release of glutamate was calcium -dependent. D-aspartate. which exchanges glutamate...out of the cytoplasmic pool. virtually eliminated the calcium -independent component of glutamate release. This synaptosomal preparation will be useful...investigation of their presynaptic mechanisms ol action. l" Hippocampus Mossy fiber expansions Synaptosomes Glutamate Dynorphin Peptides Opioids Release Calcium

Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation

PubMed Central

Alfonso, A; Cabado, A G; Vieytes, M R; Botana, L M

2000-01-01

The aim of this work was to study the relationship between intracellular alkalinization, calcium fluxes and histamine release in rat mast cells. Intracellular alkalinization was induced by nigericin, a monovalent cation ionophore, and by NH4Cl (ammonium chloride). Calcium cytosolic and intracellular pH were measured by fluorescence digital imaging using Fura-2-AM and BCECF-AM.In rat mast cells, nigericin and NH4Cl induce a dose-dependent intracellular alkalinization, a dose-dependent increase in intracellular calcium levels by releasing calcium from intracellular pools, and an activation of capacitative calcium influx.The increase in both intracellular calcium and pH activates exocytosis (histamine release) in the absence of external calcium. Under the same conditions, thapsigargin does not activate exocytosis, the main difference being that thapsigargin does not alkalinize the cytosol.After alkalinization, histamine release is intracellular-calcium dependent. With 2.5 mM EGTA and thapsigargin the cell response decreases by 62%.The cytosolic alkalinization, in addition to the calcium increase it is enough signal to elicit the exocytotic process in rat mast cells. PMID:10952669

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 inactivatesmore » 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.« less

Evaluation of calcium ion, hydroxyl ion release and pH levels in various calcium hydroxide based intracanal medicaments: An in vitro study

PubMed Central

Fulzele, Punit; Baliga, Sudhindra; Thosar, Nilima; Pradhan, Debaprya

2011-01-01

Aims: Evaluation of calcium ion and hydroxyl ion release and pH levels in various calcium hydroxide based intracanal medicaments. Objective: The purpose of this study was to evaluate calcium and hydroxyl ion release and pH levels of calcium hydroxide based products, namely, RC Cal, Metapex, calcium hydroxide with distilled water, along with the new gutta-percha points with calcium hydroxide. Materials and Methods: The materials were inserted in polyethylene tubes and immersed in deionized water. The pH variation, Ca++ and OH- release were monitored periodically for 1 week. Statistical Analysis Used: Statistical analysis was carried out using one-way analysis of variance and Tukey's post hoc tests with PASW Statistics version 18 software to compare the statistical difference. Results: After 1 week, calcium hydroxide with distilled water and RC Cal raised the pH to 12.7 and 11.8, respectively, while a small change was observed for Metapex, calcium hydroxide gutta-percha points. The calcium released after 1 week was 15.36 mg/dL from RC Cal, followed by 13.04, 1.296, 3.064 mg/dL from calcium hydroxide with sterile water, Metapex and calcium hydroxide gutta-percha points, respectively. Conclusions: Calcium hydroxide with sterile water and RC Cal pastes liberate significantly more calcium and hydroxyl ions and raise the pH higher than Metapex and calcium hydroxidegutta-percha points. PMID:22346155

Effect of degree of esterification of pectin and calcium amount on drug release from pectin-based matrix tablets.

PubMed

Sungthongjeen, Srisagul; Sriamornsak, Pornsak; Pitaksuteepong, Tasana; Somsiri, Atawit; Puttipipatkhachorn, Satit

2004-02-12

The aim of this work was to assess the effect of 2 formulation variables, the pectin type (with different degrees of esterification [DEs]) and the amount of calcium, on drug release from pectin-based matrix tablets. Pectin matrix tablets were prepared by blending indomethacin (a model drug), pectin powder, and various amounts of calcium acetate and then tableting by automatic hydraulic press machine. Differential scanning calorimetry, powder x-ray diffraction, and Fourier transformed-infrared spectroscopy studies of the compressed tablets revealed no drug-polymer interaction and the existence of drug with low crystallinity. The in-vitro release studies in phosphate buffer (United States Pharmacopeia) and tris buffer indicated that the lower the DE, the greater the time for 50% of drug release (T50). This finding is probably because of the increased binding capacity of pectin to calcium. However, when the calcium was excluded, the pectins with different DEs showed similar release pattern with insignificant difference of T50. When the amount of calcium acetate was increased from 0 to 12 mg/tablet, the drug release was significantly slower. However, a large amount of added calcium (ie, 24 mg/tablet) produced greater drug release because of the partial disintegration of tablets. The results were more pronounced in phosphate buffer, where the phosphate ions induced the precipitation of calcium phosphate. In conclusion, both pectin type and added calcium affect the drug release from the pectin-based matrix tablets.

Red wine polyphenols increase calcium in bovine aortic endothelial cells: a basis to elucidate signalling pathways leading to nitric oxide production

PubMed Central

Martin, Sophie; Andriambeloson, Emile; Takeda, Ken; Andriantsitohaina, Ramaroson

2002-01-01

The present study investigates the mechanisms by which polyphenolic compounds from red wine elicit Ca2+ mobilization in bovine aortic endothelial cells (BAECs). Two polyphenol-containing red wine extracts, red wine polyphenolic compounds (RWPC) and Provinols™, and delphinidin, an anthocyanin were used. RWPC stimulated a Ca2+-dependent release of nitric oxide (NO) from BAECs accounting for the relaxation of endothelium-denuded rat aortic rings as shown by cascade bioassay. RWPC, Provinols™ and delphinidin increased cytosolic free calcium ([Ca2+]i), by releasing Ca2+ from intracellular stores and by increasing Ca2+ entry. The RWPC-induced increase in [Ca2+]i was decreased by exposure to ryanodine (30 μM), whereas Provinols™ and delphinidin-induced increases in [Ca2+]i were decreased by bradykinin (0.1 μM) and thapsigargin (1 μM) pre-treatment. RWPC, Provinols™ and delphinidin-induced increases in [Ca2+]i were sensitive to inhibitors of phospholipase C (neomycin, 3 mM; U73122, 3 μM) and tyrosine kinase (herbimycin A, 1 μM). RWPC, Provinols™ and delphinidin induced herbimycin A (1 μM)-sensitive tyrosine phosphorylation of several intracellular proteins. Provinols™ released Ca2+ via both a cholera (CTX) and pertussis toxins (PTX)-sensitive pathway, whereas delphinidin released Ca2+ only via a PTX-sensitive mechanism. Our data contribute in defining the mechanisms of endothelial NO production caused by wine polyphenols including the increase in [Ca2+]i and the activation of tyrosine kinases. Furthermore, RWPC, Provinols™ and delphinidin display differences in the process leading to [Ca2+]i increases in endothelial cells illustrating multiple cellular targets of natural dietary polyphenolic compounds. PMID:11906973

Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents

PubMed Central

Clarke, Stephen G.; Scarnati, Matthew S.

2016-01-01

At chemical synapses, presynaptic action potentials (APs) activate voltage-gated calcium channels, allowing calcium to enter and trigger neurotransmitter release. The duration, peak amplitude, and shape of the AP falling phase alter calcium entry, which can affect neurotransmitter release significantly. In many neurons, APs do not immediately return to the resting potential, but instead exhibit a period of depolarization or hyperpolarization referred to as an afterpotential. We hypothesized that presynaptic afterpotentials should alter neurotransmitter release by affecting the electrical driving force for calcium entry and calcium channel gating. In support of this, presynaptic calcium entry is affected by afterpotentials after standard instant voltage jumps. Here, we used the mouse calyx of Held synapse, which allows simultaneous presynaptic and postsynaptic patch-clamp recording, to show that the postsynaptic response is affected significantly by presynaptic afterpotentials after voltage jumps. We therefore tested the effects of presynaptic afterpotentials using simultaneous presynaptic and postsynaptic recordings and AP waveforms or real APs. Surprisingly, presynaptic afterpotentials after AP stimuli did not alter calcium channel responses or neurotransmitter release appreciably. We show that the AP repolarization time course causes afterpotential-induced changes in calcium driving force and changes in calcium channel gating to effectively cancel each other out. This mechanism, in which electrical driving force is balanced by channel gating, prevents changes in calcium influx from occurring at the end of the AP and therefore acts to stabilize synaptic transmission. In addition, this mechanism can act to stabilize neurotransmitter release when the presynaptic resting potential changes. SIGNIFICANCE STATEMENT The shape of presynaptic action potentials (APs), particularly the falling phase, affects calcium entry and small changes in calcium influx can produce large changes in postsynaptic responses. We hypothesized that afterpotentials, which often follow APs, affect calcium entry and neurotransmitter release. We tested this in calyx of Held nerve terminals, which allow simultaneous recording of presynaptic calcium currents and postsynaptic responses. Surprisingly, presynaptic afterpotentials did not alter calcium current or neurotransmitter release. We show that the AP falling phase causes afterpotential-induced changes in electrical driving force and calcium channel gating to cancel each other out. This mechanism regulates calcium entry at the end of APs and therefore stabilizes synaptic transmission. This also stabilizes responses when the presynaptic resting potential changes. PMID:27911759

Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents.

PubMed

Clarke, Stephen G; Scarnati, Matthew S; Paradiso, Kenneth G

2016-11-09

At chemical synapses, presynaptic action potentials (APs) activate voltage-gated calcium channels, allowing calcium to enter and trigger neurotransmitter release. The duration, peak amplitude, and shape of the AP falling phase alter calcium entry, which can affect neurotransmitter release significantly. In many neurons, APs do not immediately return to the resting potential, but instead exhibit a period of depolarization or hyperpolarization referred to as an afterpotential. We hypothesized that presynaptic afterpotentials should alter neurotransmitter release by affecting the electrical driving force for calcium entry and calcium channel gating. In support of this, presynaptic calcium entry is affected by afterpotentials after standard instant voltage jumps. Here, we used the mouse calyx of Held synapse, which allows simultaneous presynaptic and postsynaptic patch-clamp recording, to show that the postsynaptic response is affected significantly by presynaptic afterpotentials after voltage jumps. We therefore tested the effects of presynaptic afterpotentials using simultaneous presynaptic and postsynaptic recordings and AP waveforms or real APs. Surprisingly, presynaptic afterpotentials after AP stimuli did not alter calcium channel responses or neurotransmitter release appreciably. We show that the AP repolarization time course causes afterpotential-induced changes in calcium driving force and changes in calcium channel gating to effectively cancel each other out. This mechanism, in which electrical driving force is balanced by channel gating, prevents changes in calcium influx from occurring at the end of the AP and therefore acts to stabilize synaptic transmission. In addition, this mechanism can act to stabilize neurotransmitter release when the presynaptic resting potential changes. The shape of presynaptic action potentials (APs), particularly the falling phase, affects calcium entry and small changes in calcium influx can produce large changes in postsynaptic responses. We hypothesized that afterpotentials, which often follow APs, affect calcium entry and neurotransmitter release. We tested this in calyx of Held nerve terminals, which allow simultaneous recording of presynaptic calcium currents and postsynaptic responses. Surprisingly, presynaptic afterpotentials did not alter calcium current or neurotransmitter release. We show that the AP falling phase causes afterpotential-induced changes in electrical driving force and calcium channel gating to cancel each other out. This mechanism regulates calcium entry at the end of APs and therefore stabilizes synaptic transmission. This also stabilizes responses when the presynaptic resting potential changes. Copyright © 2016 the authors 0270-6474/16/3611559-14$15.00/0.

Calmodulin Activation by Calcium Transients in the Postsynaptic Density of Dendritic Spines

PubMed Central

Keller, Daniel X.; Franks, Kevin M.; Bartol, Thomas M.; Sejnowski, Terrence J.

2008-01-01

The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM) and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways. PMID:18446197

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

PubMed

Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

2013-01-01

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Coenzyme Q10 inhibits the release of glutamate in rat cerebrocortical nerve terminals by suppression of voltage-dependent calcium influx and mitogen-activated protein kinase signaling pathway.

PubMed

Chang, Yi; Huang, Shu-Kuei; Wang, Su-Jane

2012-12-05

This study investigates the effects and possible mechanism of coenzyme Q10 (CoQ10) on endogenous glutamate release in the cerebral cortex nerve terminals of rats. CoQ10 inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP). CoQ10 reduced the depolarization-induced increase in cytosolic [Ca2+]c but did not alter the 4-AP-mediated depolarization. The effect of CoQ10 on evoked glutamate release was abolished by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels and mitogen-activated protein kinase kinase (MEK). In addition, CoQ10 decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK. Moreover, the inhibition of glutamate release by CoQ10 was strongly attenuated in mice without synapsin I. These results suggest that CoQ10 inhibits glutamate release from cortical synaptosomes in rats through the suppression of the presynaptic voltage-dependent Ca2+ entry and ERK/synapsin I signaling pathway.

Two-pore channels: Regulation by NAADP and customized roles in triggering calcium signals

PubMed Central

Patel, Sandip; Marchant, Jonathan; Brailoiu, Eugen

2010-01-01

NAADP is a potent regulator of cytosolic calcium levels. Much evidence suggests that NAADP activates a novel channel located on an acidic (lysosomal-like) calcium store, the mobilisation of which results in further calcium release from the endoplasmic reticulum. Here, we discuss the recent identification of a family of poorly characterized ion channels (the two-pore channels) as endo-lysosomal NAADP receptors. The generation of calcium signals by these channels is likened to those evoked by depolarisation during excitation-contraction coupling in muscle. We discuss the idea that two pore-channels can mediate a trigger release of calcium which is then amplified by calcium-induced calcium release from the endoplasmic reticulum. This is similar to the activation of voltage-sensitive calcium channels and subsequent mobilisation of sarcoplasmic reticulum calcium stores in cardiac tissue. We suggest that two-pore channels may physically interact with ryanodine receptors to account for more direct release of calcium from the endoplasmic reticulum in analogy with the conformational coupling of voltage-sensitive calcium channels and ryanodine receptors in skeletal muscle. Interaction of two-pore channels with other calcium release channels likely occurs between stores “trans-chatter” and possibly within the same store “cis-chatter”. We also speculate that trafficking of two-pore channels through the endolysosomal system facilitates interactions with calcium entry channels. Strategic placing of two-pore channels thus provides a versatile means of generating spatiotemporally complex cellular calcium signals. PMID:20621760

Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

PubMed Central

1996-01-01

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltage-dependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating models of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels. PMID:8882865

Porcine malignant hyperthermia susceptibility: hypersensitive calcium-release mechanism of skeletal muscle sarcoplasmic reticulum.

PubMed Central

O'Brien, P J

1986-01-01

This study tested the hypothesis that calcium-release from sarcoplasmic reticulum isolated from malignant hyperthermia swine had abnormal concentration-dependency on release modulators. Halothane stimulated half-maximal calcium-release at similar concentrations for malignant hyperthermia and control sarcoplasmic reticulum (0.10 +/- 0.04 mM). However, concentrations causing half-maximal calcium-release were lower for malignant hyperthermia sarcoplasmic reticulum (P less than 0.001) by an order of magnitude for Ca2+ (28.1 +/- 8.3 versus 1.23 +/- 0.45 nM), adenosine triphosphate (0.33 +/- 0.09 versus 0.023 +/- 0.014 mM) and caffeine (7.79 +/- 1.56 versus 0.80 +/- 0.44 mM). Half-maximal inhibition by Mg2+ occurred at threefold higher concentrations for malignant hyperthermia sarcoplasmic reticulum (0.23 +/- 0.02 versus 0.78 +/- 0.17 mM). The Ca2+-sensitivity curves for calcium-release by sarcoplasmic reticulum isolated from heterozygotes for the malignant hyperthermia-defect were indistinguishable from the averages of the curves for controls and malignant hyperthermia-homozygotes. Results of this study suggest that malignant hyperthermia is initiated due to a hypersensitive calcium-release mechanism which is inherited in an autosomal, codominant pattern and may be diagnosed using calcium-release sensitivity-tests on isolated sarcoplasmic reticulum. Images Fig. 1. PMID:3742367

The Epithelial Cell-derived Atopic Dermatitis Cytokine TSLP Activates Neurons to Induce Itch

PubMed Central

Wilson, Sarah R.; Thé, Lydia; Batia, Lyn M.; Beattie, Katherine; Katibah, George E.; McClain, Shannan P.; Pellegrino, Maurizio; Estandian, Daniel M.; Bautista, Diana M.

2014-01-01

Summary Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine Thymic Stromal Lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a new model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways. PMID:24094650

Involvement of the cervical sympathetic nervous system in the changes of calcium homeostasis during turpentine oil-induced stress in rats.

PubMed

Stern, J E; Ladizesky, M G; Keller Sarmiento, M I; Cardinali, D P

1993-03-01

Hypocalcemia is a common finding during stress. The objective of this study was to examine: (a) the changes in circulating calcium, parathyroid hormone (PTH) and calcitonin (CT) concentration in rats stressed by being given a subcutaneous injection of turpentine oil, and (b) the involvement of the sympathetic cervical pathway in stress-induced changes of calcium homeostasis. Four hours after receiving turpentine oil or vehicle, rats were subjected either to hypocalcemia, by being given EDTA intraperitoneally, or to hypercalcemia, by being injected CaCl2 intraperitoneally. Significant changes in serum calcium (10% decrease), serum PTH (28% increase) and CT levels (40% decrease) were observed in stressed rats. EDTA administration brought about a significantly greater hypocalcemia, and a higher PTH secretory response in turpentine oil-stressed rats. During stress, the increase of serum calcium after CaCl2 was significantly smaller, and the rise of CT was greater than in controls. In the case of CT the changes were still observed in rats subjected to superior cervical ganglionectomy (SCGx) 14 days earlier. In the case of PTH, the increase found in stressed rats, but not the augmented response after EDTA, was blunted by SCGx. The potentiation of hypocalcemia brought about by turpentine oil was no longer observed in SCGx rats. In vehicle-treated controls, SCGx delayed PTH response to hypocalcemia, but did not affect the increased response of CT to CaCl2 challenge. The results indicate that a number of changes in calcium homeostasis arise during turpentine oil stress in rats. SCGx was effective to modify the set point for PTH release, but played a minor role in affecting the augmentation of CT release during stress.

Calcium-Responsive Liposomes via a Synthetic Lipid Switch.

PubMed

Lou, Jinchao; Carr, Adam J; Watson, Alexa J; Mattern-Schain, Samuel I; Best, Michael D

2018-03-07

Liposomal drug delivery would benefit from enhanced control over content release. Here, we report a novel avenue for triggering release driven by chemical composition using liposomes sensitized to calcium-a target chosen due to its key roles in biology and disease. To demonstrate this principle, we synthesized calcium-responsive lipid switch 1, designed to undergo conformational changes upon calcium binding. The conformational change perturbs membrane integrity, thereby promoting cargo release. This was shown through fluorescence-based release assays via dose-dependent response depending on the percentage of 1 in liposomes, with minimal background leakage in controls. DLS experiments indicated dramatic changes in particle size upon treatment of liposomes containing 1 with calcium. In a comparison of ten naturally occurring metal cations, calcium provided the greatest release. Finally, STEM images showed significant changes in liposome morphology upon treatment of liposomes containing 1 with calcium. These results showcase lipid switches driven by molecular recognition principles as an exciting avenue for controlling membrane properties. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-11-15

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

PubMed

Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

2011-01-01

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

Genetically Encoded Calcium Indicators For Studying Long-Term Calcium Dynamics During Apoptosis

PubMed Central

Garcia, M. Iveth; Chen, Jessica J.; Boehning, Darren

2017-01-01

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis. PMID:28073595

Neuronal calcium sensor synaptotagmin-9 is not involved in the regulation of glucose homeostasis or insulin secretion.

PubMed

Gustavsson, Natalia; Wang, Xiaorui; Wang, Yue; Seah, Tingting; Xu, Jun; Radda, George K; Südhof, Thomas C; Han, Weiping

2010-11-09

Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7. In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice. Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic β-cells.

Floating dosage forms to prolong gastro-retention--the characterisation of calcium alginate beads.

PubMed

Stops, Frances; Fell, John T; Collett, John H; Martini, Luigi G

2008-02-28

Floating calcium alginate beads, designed to improve drug bioavailability from oral preparations compared with that from many commercially available and modified release products, have been investigated as a possible gastro-retentive dosage form. A model drug, riboflavin, was also incorporated into the formula. The aims of the current work were (a) to obtain information regarding the structure, floating ability and changes that occurred when the dosage form was placed in aqueous media, (b) to investigate riboflavin release from the calcium alginate beads in physiologically relevant media prior to in vivo investigations. Physical properties of the calcium alginate beads were investigated. Using SEM and ESEM, externally the calcium alginate beads were spherical in shape, and internally, air filled cavities were present thereby enabling floatation of the beads. The calcium alginate beads remained buoyant for times in excess of 13h, and the density of the calcium alginate beads was <1.000gcm(-3). Riboflavin release from the calcium alginate beads showed that riboflavin release was slow in acidic media, whilst in more alkali media, riboflavin release was more rapid. The characterisation studies showed that the calcium alginate beads could be considered as a potential gastro-retentive dosage form.

Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves.

PubMed

Haas, Brigitte; Schipke, Carola G; Peters, Oliver; Söhl, Goran; Willecke, Klaus; Kettenmann, Helmut

2006-02-01

In the corpus callosum, astrocytic calcium waves propagate via a mechanism involving ATP-release but not gap junctional coupling. In the present study, we report for the neocortex that calcium wave propagation depends on functional astrocytic gap junctions but is still accompanied by ATP-release. In acute slices obtained from the neocortex of mice deficient for astrocytic expression of connexin43, the calcium wave did not propagate. In contrast, in the corpus callosum and hippocampus of these mice, the wave propagated as in control animals. In addition to calcium wave propagation in astrocytes, ATP-release was recorded as a calcium signal from 'sniffer cells', a cell line expressing high-affinity purinergic receptors placed on the surface of the slice. The astrocyte calcium wave in the neocortex was accompanied by calcium signals in the 'sniffer cell' population. In the connexin43-deficient mice we recorded calcium signals from sniffer cells also in the absence of an astrocytic calcium wave. Our findings indicate that astrocytes propagate calcium signals by two separate mechanisms depending on the brain region and that ATP release can propagate within the neocortex independent from calcium waves.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

PubMed

Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

2014-03-01

Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Ozone (O{sub 3}) elicits neurotoxicity in spinal cord neurons (SCNs) by inducing ER Ca{sup 2+} release and activating the CaMKII/MAPK signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yun; Lin, Xiaowen; Zhao, XueJun

Ozone (O{sub 3}) is widely used in the treatment of spinal cord related diseases. Excess or accumulation of this photochemical air can however be neurotoxic. In this study, in vitro cultured Wister rat spinal cord neurons (SCNs) were used to investigate the detrimental effects and underlying mechanisms of O{sub 3}. Ozone in a dose-dependent manner inhibited cell viability at a range of 20 to 500 μg/ml, with the dose at 40 μg/ml resulting in a decrease of cell viability to 75%. The cell death after O{sub 3} exposure was related to endoplasmic reticulum (ER) calcium (Ca{sup 2+}) release. Intracellular Ca{supmore » 2+} chelator, ER stabilizer (inositol 1,4,5-trisphosphate receptor (IP3R) antagonist and ryanodine receptor (RyR) antagonist) and calcium/calmodulin-dependent protein kinase II (CaMKII) antagonist could effectively block Ca{sup 2+} mobilization and inhibit cell death following 40 μg/ml O{sub 3} exposure. In addition, ER Ca{sup 2+} release due to O{sub 3} exposure enhanced phospho-p38 and phospho-JNK levels and apoptosis of SCNs through activating CaMKII. Based on these results, we confirm that ozone elicits neurotoxicity in SCNs via inducing ER Ca{sup 2+} release and activating CaMKII/MAPK signaling pathway. Therefore, physicians should get attention to the selection of treatment concentrations of oxygen/ozone. And, approaches, such as chelating intracellular Ca{sup 2+} and stabilizing neuronal Ca{sup 2+} homeostasis could effectively ameliorate the neurotoxicity of O{sub 3}. - Highlights: • Exposure to O{sub 3} can reduce the viability of SCNs and cause the cell death. • Exposure to O{sub 3} can trigger RyR and IP3R dependent intracellular Ca{sup 2+} release. • Exposure to O{sub 3} can enhance the phospho-CaMKII, phospho-JNK and phospho-p38 levels.« less

Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death.

PubMed

Marks, A R

2001-04-01

Calcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+ release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+ homeostasis in the heart is beginning to emerge. The intracellular Ca2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P(o)). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

The effects of thermal stimuli on intracellular calcium change and histamine releases in rat basophilic leukemia mast cells

NASA Astrophysics Data System (ADS)

Wu, Zu-Hui; Zhu, Dan; Chen, Ji-Yao; Zhou, Lu-Wei

2012-05-01

The effects of thermal stimuli on rat basophilic leukemia mast cells were studied. The cells in calcium-contained or calcium-free buffers were thermally stimulated in the temperature range of 25-60 °C. The corresponding calcium ion concentration in cells [Ca2+]i as well as the released histamine from cells was measured with fluorescence staining methods. The ruthenium red (RR), a block of membrane calcium channels (transient receptor potential family V (TRPV)), was used in experiments. Under the stimulus of 25-50 °C, no significant difference on [Ca2+]i was found between these three groups of the cells in calcium-contained buffer without or with RR and cells in calcium-free saline, indicating that the increased calcium in cytosol did not result from the extracellular buffer but came from the intracellular calcium stores. The [Ca2+]i continuously increased under the temperature of 50-60 °C, but the RR and calcium-free saline can obviously diminish the [Ca2+]i increase at these high temperatures, reflecting that the opening of the TRPV2 channels leads to a calcium influx resulting in the [Ca2+]i increment. The histamine release also became significant in these cases. Since the released histamine is a well-known mediator for the microcirculation promotion, the histamine release from mast cells could be one of the mechanisms of thermal therapy.

Lysosomes Integrate Metabolic-Inflammatory Cross-talk in Primary Macrophage Inflammasome Activation*

PubMed Central

Weber, Kassandra; Schilling, Joel D.

2014-01-01

Macrophage dysfunction and inflammasome activation have been implicated in the pathogenesis of diabetes and its complications. Prolonged inflammation and impaired healing are hallmarks of the diabetic response to tissue injury, and excessive inflammasome activation has been associated in these phenotypes. However, the mechanisms that regulate the inflammasome in response to lipid metabolic and inflammatory stress are incompletely understood. We have shown previously that IL-1β secretion is induced in primary macrophages exposed to the dietary saturated fatty acid palmitate in combination with LPS. In this study, we sought to unravel the mechanisms underlying the activation of this lipotoxic inflammasome. We demonstrate that palmitate-loaded primary macrophages challenged with LPS activate the NLRP3 inflammasome through a mechanism that involves the lysosome. Interestingly, the lysosome was involved in both the regulation of pro-IL-1β levels and its subsequent cleavage/release. The lysosomal protease cathepsin B was required for IL-1β release but not pro-IL-1β production. In contrast, disrupting lysosomal calcium regulation decreased IL-1β release by reducing pro-IL-1β levels. The calcium pathway involved the calcium-activated phosphatase calcineurin, which stabilized IL-1β mRNA. Our findings provide evidence that the lysosome plays a key role in both the priming and assembly phases of the lipostoxic inflammasome. These findings have potential relevance to the hyperinflammatory phenotypes observed in diabetics during tissue damage or infection and identify lysosomes and calcineurin as potential therapeutic targets. PMID:24532802

Redox regulation of neuronal voltage-gated calcium channels.

PubMed

Todorovic, Slobodan M; Jevtovic-Todorovic, Vesna

2014-08-20

Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain.

Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis.

PubMed

Kyrmizi, Irene; Ferreira, Helena; Carvalho, Agostinho; Figueroa, Julio Alberto Landero; Zarmpas, Pavlos; Cunha, Cristina; Akoumianaki, Tonia; Stylianou, Kostas; Deepe, George S; Samonis, George; Lacerda, João F; Campos, António; Kontoyiannis, Dimitrios P; Mihalopoulos, Nikolaos; Kwon-Chung, Kyung J; El-Benna, Jamel; Valsecchi, Isabel; Beauvais, Anne; Brakhage, Axel A; Neves, Nuno M; Latge, Jean-Paul; Chamilos, Georgios

2018-05-30

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca 2+ signalling pathway that depends on intracellular Ca 2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca 2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca 2+ -CaM signalling in aspergillosis. Finally, we demonstrate that Ca 2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca 2+ -CaM signalling to inhibit LAP. These findings reveal the important role of Ca 2+ -CaM signalling in antifungal immunity and identify an immunological function of Ca 2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.

Calcium-containing phosphopeptides pave the secretory pathway for efficient protein traffic and secretion in fungi.

PubMed

Martín, Juan F

2014-09-10

Casein phosphopeptides (CPPs) containing chelated calcium drastically increase the secretion of extracellular homologous and heterologous proteins in filamentous fungi. Casein phosphopeptides released by digestion of alpha - and beta-casein are rich in phosphoserine residues (SerP). They stimulate enzyme secretion in the gastrointestinal tract and enhance the immune response in mammals, and are used as food supplements. It is well known that casein phosphopeptides transport Ca2+ across the membranes and play an important role in Ca2+ homeostasis in the cells. Addition of CPPs drastically increases the production of heterologous proteins in Aspergillus as host for industrial enzyme production. Recent proteomics studies showed that CPPs alter drastically the vesicle-mediated secretory pathway in filamentous fungi, apparently because they change the calcium concentration in organelles that act as calcium reservoirs. In the organelles calcium homeostasis a major role is played by the pmr1 gene, that encodes a Ca2+/Mn2+ transport ATPase, localized in the Golgi complex; this transporter controls the balance between intra-Golgi and cytoplasmic Ca2+ concentrations. A Golgi-located casein kinase (CkiA) governs the ER to Golgi directionality of the movement of secretory proteins by interacting with the COPII coat of secretory vesicles when they reach the Golgi. Mutants defective in the casein-2 kinase CkiA show abnormal targeting of some secretory proteins, including cytoplasmic membrane amino acid transporters that in ckiA mutants are miss-targeted to vacuolar membranes. Interestingly, addition of CPPs increases a glyceraldehyde-3-phpshate dehydrogenase protein that is known to associate with microtubules and act as a vesicle/membrane fusogenic agent. In summary, CPPs alter the protein secretory pathway in fungi adapting it to a deregulated protein traffic through the organelles and vesicles what results in a drastic increase in secretion of heterologous and also of some homologous proteins.

Calcium modified edible Canna (Canna edulis L) starch for controlled released matrix

NASA Astrophysics Data System (ADS)

Putri, A. P.; Ridwan, M.; Darmawan, T. A.; Darusman, F.; Gadri, A.

2017-07-01

Canna edulis L starch was modified with calcium chloride in order to form controlled released matrix. Present study aim to analyze modified starch characteristic. Four different formulation of ondansetron granules was used to provide dissolution profile of controlled released, two formula consisted of 15% and 30% modified starch, one formula utilized matrix reference standards and the last granules was negative control. Methocel-hydroxypropyl methyl cellulose was used as controlled released matrix reference standards in the third formula. Calcium starch was synthesized in the presence of sodium hydroxide to form gelatinized mass and calcium chloride as the cross linking agent. Physicochemical and dissolution properties of modified starch for controlled released application were investigated. Modified starch has higher swelling index, water solubility and compressibility index. Three of four different formulation of granules provide dissolution profile of controlled released. The profiles indicate granules which employed calcium Canna edulis L starch as matrix are able to resemble controlled drug released profile of matrix reference, however their bigger detain ability lead to lower bioavailability.

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore.

PubMed

Rottenberg, Hagai; Hoek, Jan B

2017-10-01

Excessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging-related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro-apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Main pathways of action of Brazilian red propolis on the modulation of neutrophils migration in the inflammatory process.

PubMed

Bueno-Silva, Bruno; Franchin, Marcelo; Alves, Claudiney de Freitas; Denny, Carina; Colón, David Fernando; Cunha, Thiago Mattar; Alencar, Severino Matias; Napimoga, Marcelo Henrique; Rosalen, Pedro Luiz

2016-12-01

Brazilian propolis is popularly used as treatment for different diseases including the ones with inflammatory origin. Brazilian red propolis chemical profile and its anti-inflammatory properties were recently described however, its mechanism of action has not been investigated yet. Elucidate Brazilian red propolis major pathways of action on the modulation of neutrophil migration during the inflammatory process. The ethanolic extract of propolis (EEP) activity was investigated for neutrophil migration into the peritoneal cavity, intravital microscopy (rolling and adhesion of leukocytes), quantification of cytokines TNF-α, IL-1β and chemokines CXCL1/KC, CXCL2/MIP-2, neutrophil chemotaxis induced by CXCL2/MIP-2, calcium influx and CXCR2 expression on neutrophils. EEP at 10mg/kg prevented neutrophil migration into peritoneal cavity (p < 0.05), reduced leukocyte rolling and adhesion on the mesenteric microcirculation (p < 0.05) and inhibited the release TNF-α, IL-1β, CXCL1/KC and CXCL2/MIP-2 (p < 0.05). EEP at 0.01, 0.1 and 1µg/ml reduced the CXCL2/MIP-2-induced neutrophils chemotaxis (p < 0.05) without affect cell viability (p > 0.05).EEP at 1µg/ml decreased the calcium influx induced by CXCL2/MIP-2 (p<0.05). On the other hand, none of EEP concentrations tested altered CXCR2 expression by neutrophils (p>0.05). Brazilian red propolis appears as a promising anti-inflammatory natural product which mechanism seems to be by reducing leukocyte rolling and adhesion; TNF-α, IL-1β, CXCL1/KC and CXCL2/MIP-2 release; CXCL2/MIP-2-induced chemotaxis and calcium influx. Copyright © 2016 Elsevier GmbH. All rights reserved.

Mechanisms of Nattokinase in protection of cerebral ischemia.

PubMed

Ji, Hongrui; Yu, Liang; Liu, Keyu; Yu, Zhigang; Zhang, Qian; Zou, Fengjuan; Liu, Bo

2014-12-15

In vivo, the level of cyclic Adenosine Monophosphate (cAMP) and the pathway of the Janus Kinase1/Signal Transducers and Activators of Transcription1 (JAK1/STAT1) were studied. In vitro, the Ca(2+) mobilization in human platelet stimulated by thrombin was observed. In addition, vasomotion of vascular smooth muscle was measured by adding KCl or norepinephrine(NE) under the Ca(2+) contained bath solutions. The effect induced by NE in the presence of N-nitro-L-arginine methyl ester (L-NAME) or indometacin (Indo) was also detected. At last, the levels of tissue plasminogen activator (t-PA) and Plasminogen activator inhibitor-1 (PAI-1) in cultured supernatans in Human umbilical vein endothelial cells (Huvecs) were measured by means of ELISA kit. Results showed that Nattokinase (NK) significantly increased the cAMP level, activated the signal passage of JAK1/STAT1 in injured part and inhibited remarkably the rise of platelet intracellular Ca(2+) ([Ca(2+)]i) in human platelet. Furthermore, NK relaxed rat thoracic aortic artery in the dose-dependent manner and in the endothelium dependent manner and its effect could be attenuated by L-NAME. Also, the secretion of t-PA and PAI-1 were reduced stimulated by Adr on Huvecs. These data indicated that the neuroprotective effect of NK was associated with its antiplatelet activity by elevating cAMP level and attenuating the calcium release from calcium stores; with its anti-apoptotic effect through the activation of JAK1/STAT1 pathway; with its relaxing vascular smooth muscle by promoting synthesis and release of NO, reducing ROC calcium ion influx and with its protection on endothelial cells through increasing fibrinolytic activity and facilitating spontaneous thrombolysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis.

PubMed

Garcia, M Iveth; Chen, Jessica J; Boehning, Darren

2017-01-01

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extracellular Calcium Has Multiple Targets to Control Cell Proliferation.

PubMed

Capiod, Thierry

2016-01-01

Calcium channels and the two G-protein coupled receptors sensing extracellular calcium, calcium-sensing receptor (CaSR) and GPRC6a, are the two main means by which extracellular calcium can signal to cells and regulate many cellular processes including cell proliferation, migration and invasion of tumoral cells. Many intracellular signaling pathways are sensitive to cytosolic calcium rises and conversely intracellular signaling pathways can modulate calcium channel expression and activity. Calcium channels are undoubtedly involved in the former while the CaSR and GPRC6a are most likely to interfere with the latter. As for neurotransmitters, calcium ions use plasma membrane channels and GPCR to trigger cytosolic free calcium concentration rises and intracellular signaling and regulatory pathways activation. Calcium sensing GPCR, CaSR and GPRC6a, allow a supplemental degree of control and as for metabotropic receptors, they not only modulate calcium channel expression but they may also control calcium-dependent K+ channels. The multiplicity of intracellular signaling pathways involved, their sensitivity to local and global intracellular calcium increase and to CaSR and GPRC6a stimulation, the presence of membrane signalplex, all this confers the cells the plasticity they need to convert the effects of extracellular calcium into complex physiological responses and therefore determine their fate.

Short-Term Facilitation at a Detonator Synapse Requires the Distinct Contribution of Multiple Types of Voltage-Gated Calcium Channels.

PubMed

Chamberland, Simon; Evstratova, Alesya; Tóth, Katalin

2017-05-10

Neuronal calcium elevations are shaped by several key parameters, including the properties, density, and the spatial location of voltage-gated calcium channels (VGCCs). These features allow presynaptic terminals to translate complex firing frequencies and tune the amount of neurotransmitter released. Although synchronous neurotransmitter release relies on both P/Q- and N-type VGCCs at hippocampal mossy fiber-CA3 synapses, the specific contribution of VGCCs to calcium dynamics, neurotransmitter release, and short-term facilitation remains unknown. Here, we used random-access two-photon calcium imaging together with electrophysiology in acute mouse hippocampal slices to dissect the roles of P/Q- and N-type VGCCs. Our results show that N-type VGCCs control glutamate release at a limited number of release sites through highly localized Ca 2+ elevations and support short-term facilitation by enhancing multivesicular release. In contrast, Ca 2+ entry via P/Q-type VGCCs promotes the recruitment of additional release sites through spatially homogeneous Ca 2+ elevations. Altogether, our results highlight the specialized contribution of P/Q- and N-types VGCCs to neurotransmitter release. SIGNIFICANCE STATEMENT In presynaptic terminals, neurotransmitter release is dynamically regulated by the transient opening of different types of voltage-gated calcium channels. Hippocampal giant mossy fiber terminals display extensive short-term facilitation during repetitive activity, with a large several fold postsynaptic response increase. Though, how giant mossy fiber terminals leverage distinct types of voltage-gated calcium channels to mediate short-term facilitation remains unexplored. Here, we find that P/Q- and N-type VGCCs generate different spatial patterns of calcium elevations in giant mossy fiber terminals and support short-term facilitation through specific participation in two mechanisms. Whereas N-type VGCCs contribute only to the synchronization of multivesicular release, P/Q-type VGCCs act through microdomain signaling to recruit additional release sites. Copyright © 2017 the authors 0270-6474/17/374913-15$15.00/0.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

PubMed

Kapustin, Alexander N; Chatrou, Martijn L L; Drozdov, Ignat; Zheng, Ying; Davidson, Sean M; Soong, Daniel; Furmanik, Malgorzata; Sanchis, Pilar; De Rosales, Rafael Torres Martin; Alvarez-Hernandez, Daniel; Shroff, Rukshana; Yin, Xiaoke; Muller, Karin; Skepper, Jeremy N; Mayr, Manuel; Reutelingsperger, Chris P; Chester, Adrian; Bertazzo, Sergio; Schurgers, Leon J; Shanahan, Catherine M

2015-04-10

Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention. © 2015 American Heart Association, Inc.

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

PubMed Central

Torres-Atencio, Ivonne; Ainsua-Enrich, Erola; de Mora, Fernando; Picado, César; Martín, Margarita

2014-01-01

Background Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition. PMID:25329458

Cornuside inhibits mast cell-mediated allergic response by down-regulating MAPK and NF-κB signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Liangchang; Jin, Guangyu; Jiang, Jingzhi

Aims: The present study is to investigate the effect of cornuside on mast cell-mediated allergic response, as well as its possible mechanisms of action. Methods: To test the anti-allergic effects of cornuside in vivo, local extravasation was induced by local injection of anti-dinitrophenyl immunoglobulin E (IgE) followed by intravenous antigenic challenge in passive cutaneous anaphylaxis model rats. Mast cell viability was determined using MTT assay. Histamine content from rat peritoneal mast cells was measured by the radioenzymatic method. To investigate the mechanisms by which cornuside affects the reduction of histamine release, the levels of calcium uptake were measured. To examine whethermore » cornuside affects the expression of pro-inflammatory cytokines, Western blotting and ELISA were carried out. Results: Oral administration of cornuside inhibited passive cutaneous anaphylaxis in rats. Presence of cornuside attenuated IgE-induced histamine release from rat peritoneal mast cells. The inhibitory effect of cornuside on histamine release was mediated by the modulation of intracellular calcium. In addition, cornuside decreased phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated production and secretion of pro-inflammatory cytokines such as TNF-α and IL-6 in human mast cells. The inhibitory effect of cornuside on pro-inflammatory cytokines was dependent on nuclear factor-κB and p38 mitogen-activated protein kinase. Conclusions: The present study provides evidence that cornuside inhibits mast cell-derived inflammatory allergic reactions by blocking histamine release and pro-inflammatory cytokine expression. Furthermore, in vivo and in vitro anti-allergic effects of cornuside suggest a possible therapeutic application of this agent in inflammatory allergic diseases.« less

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

PubMed

Macková, Katarina; Zahradníková, Alexandra; Hoťka, Matej; Hoffmannová, Barbora; Zahradník, Ivan; Zahradníková, Alexandra

2017-12-01

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

The Orai-1 and STIM-1 Complex Controls Human Dendritic Cell Maturation

PubMed Central

Félix, Romain; Crottès, David; Delalande, Anthony; Fauconnier, Jérémy; Lebranchu, Yvon; Le Guennec, Jean-Yves; Velge-Roussel, Florence

2013-01-01

Ca2+ signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca2+ signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca2+ stores that results in a store-operated Ca2+ entry (SOCE). This Ca2+ influx was inhibited by 2-APB and exhibited a Ca2+permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca2+ entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins. PMID:23700407

Differential regulation of GnRH secretion in the preoptic area (POA) and the median eminence (ME) in male mice.

PubMed

Glanowska, Katarzyna M; Moenter, Suzanne M

2015-01-01

GnRH release in the median eminence (ME) is the central output for control of reproduction. GnRH processes in the preoptic area (POA) also release GnRH. We examined region-specific regulation of GnRH secretion using fast-scan cyclic voltammetry to detect GnRH release in brain slices from adult male mice. Blocking endoplasmic reticulum calcium reuptake to elevate intracellular calcium evokes GnRH release in both the ME and POA. This release is action potential dependent in the ME but not the POA. Locally applied kisspeptin induced GnRH secretion in both the ME and POA. Local blockade of inositol triphospate-mediated calcium release inhibited kisspeptin-induced GnRH release in the ME, but broad blockade was required in the POA. In contrast, kisspeptin-evoked secretion in the POA was blocked by local gonadotropin-inhibitory hormone, but broad gonadotropin-inhibitory hormone application was required in the ME. Although action potentials are required for GnRH release induced by pharmacologically-increased intracellular calcium in the ME and kisspeptin-evoked release requires inositol triphosphate-mediated calcium release, blocking action potentials did not inhibit kisspeptin-induced GnRH release in the ME. Kisspeptin-induced GnRH release was suppressed after blocking both action potentials and plasma membrane Ca(2+) channels. This suggests that kisspeptin action in the ME requires both increased intracellular calcium and influx from the outside of the cell but not action potentials. Local interactions among kisspeptin and GnRH processes in the ME could thus stimulate GnRH release without involving perisomatic regions of GnRH neurons. Coupling between action potential generation and hormone release in GnRH neurons is thus likely physiologically labile and may vary with region.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed Central

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-01-01

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals. PMID:8947471

Neutrophil extracellular traps release induced by Leishmania: role of PI3Kγ, ERK, PI3Kσ, PKC, and [Ca2+

PubMed Central

DeSouza-Vieira, Thiago; Guimarães-Costa, Anderson; Rochael, Natalia C.; Lira, Maria N.; Nascimento, Michelle T.; Lima-Gomez, Phillipe de Souza; Mariante, Rafael M.; Persechini, Pedro M.; Saraiva, Elvira M.

2016-01-01

Upon in vitro stimulation, neutrophils undergo a cell death named netosis. This process is characterized by extracellular release of chromatin scaffold associated with granular and cytoplasmic proteins, which together, ensnare and kill microbes. We have previously described that interaction of Leishmania amazonensis with human neutrophils leads to the release of neutrophil extracellular traps, which trap and kill the parasite. However, the signaling leading to Leishmania induced netosis is still unknown. Thus, we sought to evaluate signaling events that drive L. amazonensis induced neutrophil extracellular trap release from human neutrophils. Here, we found that PI3K, independently of protein kinase B, has a role in parasite-induced netosis. We also described that the main isoforms involved are PI3Kγ and PI3Kδ, which work in reactive oxygen species-dependent and -independent ways, respectively. We demonstrated that activation of ERK downstream of PI3Kγ is important to trigger reactive oxygen species-dependent, parasite-induced netosis. Pharmacological inhibition of protein kinase C also significantly decreased parasite-induced neutrophil extracellular trap release. Intracellular calcium, regulated by PI3Kδ, represents an alternative reactive oxygen species-independent pathway of netosis stimulated by L. amazonensis. Finally, intracellular calcium mobilization and reactive oxygen species generation are the major regulators of parasite-induced netosis. Our results contribute to a better understanding of the signaling behind netosis induced by interactions between Leishmania and neutrophils. PMID:27154356

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

PubMed

Kaplan, Mehmet Mahsum; Sultana, Nasreen; Benedetti, Ariane; Obermair, Gerald J; Linde, Nina F; Papadopoulos, Symeon; Dayal, Anamika; Grabner, Manfred; Flucher, Bernhard E

2018-06-26

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (Ca V 1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Releasing effects in flame photometry: Determination of calcium

USGS Publications Warehouse

Dinnin, J.I.

1960-01-01

Strontium, lanthanum, neodymium, samarium, and yttrium completely release the flame emission of calcium from the depressive effects of sulfate, phosphate, and aluminate. Magnesium, beryllium, barium, and scandium release most of the calcium emission. These cations, when present in high concentration, preferentially form compounds with the depressing anions when the solution is evaporated rapidly in the flame. The mechanism of the interference and releasing effects is explained on the basis of the chemical equilibria in the evaporating droplets of solution and is shown to depend upon the nature of the compounds present in the aqueous phase of the solution. The need for background correction techniques is stressed. The releasing effect is used in the determination of calcium in silicate rocks without the need for separations.

The spontaneous and evoked release of spermine from rat brain in vitro.

PubMed Central

Harman, R. J.; Shaw, G. G.

1981-01-01

1 The efflux of previously accumulated [3H]-spermine from brain slices was measured using a continuous perfusion system. The spontaneous efflux was biphasic, consisting of an initial rapid efflux followed by a much slower release. 2 The slices were depolarized by the addition to the medium of high potassium concentrations, ouabain or veratrine. 3 At concentrations greater than 30 mM, potassium evoked a striking increase in the release of [3H]-spermine. Following uptake in the presence of 5.7 x 10(-9)M [3H]-spermine, K+-evoked release was dependent on the presence of calcium ions. Release of spermine after uptake at 5.6 x 10(-8)M or 5.0 x 10(-7)M was not calcium-dependent. 4 The calcium-dependent, K+-stimulated release of spermine was inhibited in the presence of diphenylhydantoin (5 x 10(-5)M) or ruthenium red (10(-5)M). 5 Following uptake of 5.7 x 10(-9)M [3H]-spermine in a sodium-free medium, the calcium-dependent, K+-stimulated release was significantly inhibited. 5 Ouabain (10(-4)M) caused a large but calcium-independent increase in the efflux of [3H]-spermine. 7 Veratrine-induced release was less substantial but was increased in a calcium-free medium. Release evoked by veratrine was abolished in the absence of sodium. 8 These results are discussed with respect to a possible 'neurotransmitter' or 'neuromodulator' role for spermine. PMID:6169383

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

PubMed

Adamczewski, M; Paolini, R; Kinet, J P

1992-09-05

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

PubMed Central

Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro

2017-01-01

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation. PMID:28817667

A molecular web: endoplasmic reticulum stress, inflammation, and oxidative stress.

PubMed

Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d'Hellencourt, Christian; Ravanan, Palaniyandi

2014-01-01

Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress.

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

PubMed

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.

Redox Regulation of Neuronal Voltage-Gated Calcium Channels

PubMed Central

Jevtovic-Todorovic, Vesna

2014-01-01

Abstract Significance: Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Recent Advances: Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. Critical Issues: A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Future Directions: Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain. Antioxid. Redox Signal. 21, 880–891. PMID:24161125

Sound Waves Induce Neural Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells via Ryanodine Receptor-Induced Calcium Release and Pyk2 Activation.

PubMed

Choi, Yura; Park, Jeong-Eun; Jeong, Jong Seob; Park, Jung-Keug; Kim, Jongpil; Jeon, Songhee

2016-10-01

Mesenchymal stem cells (MSCs) have shown considerable promise as an adaptable cell source for use in tissue engineering and other therapeutic applications. The aims of this study were to develop methods to test the hypothesis that human MSCs could be differentiated using sound wave stimulation alone and to find the underlying mechanism. Human bone marrow (hBM)-MSCs were stimulated with sound waves (1 kHz, 81 dB) for 7 days and the expression of neural markers were analyzed. Sound waves induced neural differentiation of hBM-MSC at 1 kHz and 81 dB but not at 1 kHz and 100 dB. To determine the signaling pathways involved in the neural differentiation of hBM-MSCs by sound wave stimulation, we examined the Pyk2 and CREB phosphorylation. Sound wave induced an increase in the phosphorylation of Pyk2 and CREB at 45 min and 90 min, respectively, in hBM-MSCs. To find out the upstream activator of Pyk2, we examined the intracellular calcium source that was released by sound wave stimulation. When we used ryanodine as a ryanodine receptor antagonist, sound wave-induced calcium release was suppressed. Moreover, pre-treatment with a Pyk2 inhibitor, PF431396, prevented the phosphorylation of Pyk2 and suppressed sound wave-induced neural differentiation in hBM-MSCs. These results suggest that specific sound wave stimulation could be used as a neural differentiation inducer of hBM-MSCs.

Vasorelaxant effect of quercetin on cerebral basilar artery in vitro and the underlying mechanisms study.

PubMed

Yuan, Tian-Yi; Niu, Zi-Ran; Chen, Di; Chen, Yu-Cai; Zhang, Hui-Fang; Fang, Lian-Hua; Du, Guan-Hua

2018-04-25

The aim of this study is to investigate the vasorelaxant effect of quercetin on cerebral basilar artery in vitro and provide a preliminary discussion concerning the underlying mechanisms. Using a DMT-isolated micro vessel system, quercetin was found to exhibit a vasodilatory effect on basilar arteries contracted by potassium chloride (KCl), endothelin-1 (ET-1), and 5-hydroxytryptamine (5-HT). The vasorelaxant effect of quercetin was partially attenuated when endothelium cells were removed. L-NAME, indomethacin, and ODQ treatment also decreased the potency of quercetin. In endothelium-denuded rings, the vasorelaxant effect of quercetin was not influenced by K + channel inhibitors. However, quercetin inhibited KCl induced extracellular calcium influx and ET-1 induced transient intracellular calcium release in a Ca 2+ -free solution. In conclusion, quercetin induced relaxation of the basilar artery in vitro is partially dependent on endothelium, which is mainly related to NO and COX pathways. It also induces relaxation through blockage of calcium channels.

Controlling the strontium-doping in calcium phosphate microcapsules through yeast-regulated biomimetic mineralization.

PubMed

Huang, Miaojun; Li, Tianjie; Pan, Ting; Zhao, Naru; Yao, Yongchang; Zhai, Zhichen; Zhou, Jiaan; Du, Chang; Wang, Yingjun

2016-10-01

Yeast cells have controllable biosorption on metallic ions during metabolism. However, few studies were dedicated to using yeast-regulated biomimetic mineralization process to control the strontium-doped positions in calcium phosphate microcapsules. In this study, the yeast cells were allowed to pre-adsorb strontium ions metabolically and then served as sacrificing template for the precipitation and calcination of mineral shell. The pre-adsorption enabled the microorganism to enrich of strontium ions into the inner part of the microcapsules, which ensured a slow-release profile of the trace element from the microcapsule. The co-culture with human marrow stromal cells showed that gene expressions of alkaline phosphatase and Collagen-I were promoted. The promotion of osteogenic differentiation was further confirmed in the 3D culture of cell-material complexes. The strategy using living microorganism as 'smart doping apparatus' to control incorporation of trace element into calcium phosphate paved a pathway to new functional materials for hard tissue regeneration.

Calcium channel blockers and transmitter release at the normal human neuromuscular junction.

PubMed

Protti, D A; Reisin, R; Mackinley, T A; Uchitel, O D

1996-05-01

Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and omega-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or omega-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.

Effect of particle size on calcium release and elevation of pH of endodontic cements.

PubMed

Saghiri, Mohammad Ali; Asatourian, Armen; Orangi, Jafar; Lotfi, Mehrdad; Soukup, Jason W; Garcia-Godoy, Franklin; Sheibani, Nader

2015-06-01

Elevation of pH and calcium ion release are of great importance in antibacterial activity and the promotion of dental soft and hard tissue healing process. In this study, we evaluated the effect of particle size on the elevation of pH and the calcium ion release from calcium silicate-based dental cements. Twelve plastic tubes were divided into three groups, filled with white mineral trioxide aggregate (WMTA), WMTA plus 1% methylcellulose, and nano-modified WMTA (nano-WMTA), and placed inside flasks containing 10 ml of distilled water. The pH values were measured using a pH sensor 3, 24, 72, and 168 h after setting of the cements. The calcium ion release was measured using an atomic absorption spectrophotometer with same sample preparation method. Data were subjected to two-way analysis of variance (anova) followed by post hoc Tukey tests with significance level of P < 0.05. Nano-WMTA showed significant pH elevation only after 24 h (P < 0.05) compared with WMTA, and after 3, 24, and 72 h compared with WMTA plus 1% methylcellulose (P < 0.05). Nano-WMTA showed significantly higher calcium ion release values compared to the other two groups (P < 0.05). Nano-modification of WMTA remarkably increased the calcium ion release at all time intervals postsetting, which can significantly influence the osteogenic properties of human dental pulp cells and as a consequence enhance mineralized matrix nodule formation to achieve desirable clinical outcomes. However, the increase in pH values mainly occurred during the short time postsetting. Addition of 1% methylcellulose imposed a delay in elevation of pH and calcium ion release by WMTA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genetic Ablation of Calcium-independent Phospholipase A2γ (iPLA2γ) Attenuates Calcium-induced Opening of the Mitochondrial Permeability Transition Pore and Resultant Cytochrome c Release*

PubMed Central

Moon, Sung Ho; Jenkins, Christopher M.; Kiebish, Michael A.; Sims, Harold F.; Mancuso, David J.; Gross, Richard W.

2012-01-01

Herein, we demonstrate that calcium-independent phospholipase A2γ (iPLA2γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA2γ−/− mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA2γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA2γ−/− mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca2+-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA2γ−/− mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA2γ−/− mouse liver were resistant to Ca2+/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA2γ−/− mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA2γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death. PMID:22778252

Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(+)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices.

PubMed

Valentijn, K; Tranchand Bunel, D; Vaudry, H

1992-07-01

The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K(+)-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd2+ concentrations and sensitivity to Ni2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.

Effects of calcium hydroxide addition on the physical and chemical properties of a calcium silicate-based sealer.

PubMed

Kuga, Milton Carlos; Duarte, Marco Antonio Hungaro; Sant'anna-Júnior, Arnaldo; Keine, Kátia Cristina; Faria, Gisele; Dantas, Andrea Abi Rached; Guiotti, Flávia Angélica

2014-06-01

Recently, various calcium silicate-based sealers have been introduced for use in root canal filling. The MTA Fillapex is one of these sealers, but some of its physicochemical properties are not in accordance with the ISO requirements. The aim of this study was to evaluate the flowability, pH level and calcium release of pure MTA Fillapex (MTAF) or containing 5% (MTAF5) or 10% (MTAF10) calcium hydroxide (CH), in weight, in comparison with AH Plus sealer. The flowability test was performed according to the ISO 6876:2001 requirements. For the pH level and calcium ion release analyses, the sealers were placed individually (n=10) in plastic tubes and immersed in deionized water. After 24 hours, 7 and 14 days, the water in which each specimen had been immersed was evaluated to determine the pH level changes and calcium released. Flowability, pH level and calcium release data were analyzed statistically by the ANOVA test (α=5%). In relation to flowability: MTAF>AH Plus>MTAF5>MTAF10. In relation to the pH level, for 24 h: MTAF5=MTAF10=MTAF>AH Plus; for 7 and 14 days: MTAF5=MTAF10>MTAF>AH Plus. For the calcium release, for all periods: MTAF>MTAF5=MTAF10>AH Plus. The addition of 5% CH to the MTA Fillapex (in weight) is an alternative to reduce the high flowability presented by the sealer, without interfering in its alkalization potential.

Release of mitochondrial glutathione and calcium by a cyclosporin A-sensitive mechanism occurs without large amplitude swelling.

PubMed

Savage, M K; Reed, D J

1994-11-15

Treatment of isolated mitochondria with calcium and inorganic phosphate induces inner membrane permeability that is thought to be mediated through a non-selective, calcium-dependent pore. The inner membrane permeability results in the rapid efflux of small matrix solutes such as glutathione and calcium, loss of coupled functions, and large amplitude swelling. We have identified conditions of permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing calcium and inorganic phosphate abolished large-amplitude swelling of mitochondria but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial glutathione and calcium. The release of both glutathione and calcium was inhibited by the addition of cyclosporin A, a potent inhibitor of permeability transition. Transmission electron microscopy analysis, combined with the glutathione and calcium release data, indicate that permeability transition can be observed in the absence of large-amplitude swelling. Permeability transition occurring both with and without large-amplitude swelling was accompanied by a collapse of the membrane potential. We conclude that cyclosporin A-sensitive permeability transition can occur without obvious morphological changes such as large-amplitude swelling. Monitoring the cyclosporin A-sensitive release of concentrated endogenous matrix solutes, such as GSH, may be a sensitive and useful indicator of permeability transition.

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

PubMed

Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

Calcium silicate-based drug delivery systems.

PubMed

Zhu, Ying-Jie; Guo, Xiao-Xuan; Sham, Tsun-Kong

2017-02-01

Compared with other inorganic materials such as silica, metal oxides, noble metals and carbon, calcium silicate-based materials, especially nanostructured calcium silicate materials, have high biocompatibility, bioactivity and biodegradability, high specific surface area, nanoporous/hollow structure, high drug-loading capacity, pH-responsive drug release behavior and desirable drug release properties, and thus they are promising for the application in drug delivery. Calcium silicate-based drug delivery systems have a long drug-release time, which can significantly prolong the therapeutic effect of drugs. Another advantage of calcium silicate-based drug delivery systems is their pH-responsive drug release property, which can act as an ideal platform for targeted drug delivery. Areas covered: In recent years, studies have been carried out on calcium silicate-based drug delivery systems, and important results and insights have been documented. This article is not intended to offer a comprehensive review on the research on calcium silicate-based drug delivery systems, but presents some examples reported in the literature, and includes new insights obtained by tracking the interactions between drug molecules and calcium silicate carriers on the molecular level using the synchrotron-based X-ray spectroscopy. Expert opinion: Finally, our opinions on calcium silicate-based drug delivery systems are provided, and several research directions for the future studies are proposed.

Neuromodulatory changes in short-term synaptic dynamics may be mediated by two distinct mechanisms of presynaptic calcium entry.

PubMed

Oh, Myongkeun; Zhao, Shunbing; Matveev, Victor; Nadim, Farzan

2012-12-01

Although synaptic output is known to be modulated by changes in presynaptic calcium channels, additional pathways for calcium entry into the presynaptic terminal, such as non-selective channels, could contribute to modulation of short term synaptic dynamics. We address this issue using computational modeling. The neuropeptide proctolin modulates the inhibitory synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron, two slow-wave bursting neurons in the pyloric network of the crab Cancer borealis. Proctolin enhances the strength of this synapse and also changes its dynamics. Whereas in control saline the synapse shows depression independent of the amplitude of the presynaptic LP signal, in proctolin, with high-amplitude presynaptic LP stimulation the synapse remains depressing while low-amplitude stimulation causes facilitation. We use simple calcium-dependent release models to explore two alternative mechanisms underlying these modulatory effects. In the first model, proctolin directly targets calcium channels by changing their activation kinetics which results in gradual accumulation of calcium with low-amplitude presynaptic stimulation, leading to facilitation. The second model uses the fact that proctolin is known to activate a non-specific cation current I ( MI ). In this model, we assume that the MI channels have some permeability to calcium, modeled to be a result of slow conformation change after binding calcium. This generates a gradual increase in calcium influx into the presynaptic terminals through the modulatory channel similar to that described in the first model. Each of these models can explain the modulation of the synapse by proctolin but with different consequences for network activity.

Trigeminal ganglion neuron subtype-specific alterations of CaV2.1 calcium current and excitability in a Cacna1a mouse model of migraine

PubMed Central

Fioretti, B; Catacuzzeno, L; Sforna, L; Gerke-Duncan, M B; van den Maagdenberg, A M J M; Franciolini, F; Connor, M; Pietrobon, D

2011-01-01

Abstract Familial hemiplegic migraine type-1 (FHM1), a monogenic subtype of migraine with aura, is caused by gain-of-function mutations in CaV2.1 (P/Q-type) calcium channels. The consequences of FHM1 mutations on the trigeminovascular pathway that generates migraine headache remain largely unexplored. Here we studied the calcium currents and excitability properties of two subpopulations of small-diameter trigeminal ganglion (TG) neurons from adult wild-type (WT) and R192Q FHM1 knockin (KI) mice: capsaicin-sensitive neurons without T-type calcium currents (CS) and capsaicin-insensitive neurons characterized by the expression of T-type calcium currents (CI-T). Small TG neurons retrogradely labelled from the dura are mostly CS neurons, while CI-T neurons were not present in the labelled population. CS and CI-T neurons express CaV2.1 channels with different activation properties, and the CaV2.1 channels are differently affected by the FHM1 mutation in the two TG neuron subtypes. In CI-T neurons from FHM1 KI mice there was a larger P/Q-type current density following mild depolarizations, a larger action potential (AP)-evoked calcium current and a longer AP duration when compared to CI-T neurons from WT mice. In striking contrast, the P/Q-type current density, voltage dependence and kinetics were not altered by the FHM1 mutation in CS neurons. The excitability properties of mutant CS neurons were also unaltered. Congruently, the FHM1 mutation did not alter depolarization-evoked CGRP release from the dura mater, while CGRP release from the trigeminal ganglion was larger in KI compared to WT mice. Our findings suggest that the facilitation of peripheral mechanisms of CGRP action, such as dural vasodilatation and nociceptor sensitization at the meninges, does not contribute to the generation of headache in FHM1. PMID:22005682

THE REGULATORY ROLE OF DIVALENT CATIONS IN HUMAN GRANULOCYTE CHEMOTAXIS

PubMed Central

Gallin, John I.; Rosenthal, Alan S.

1974-01-01

Optimal human granulocyte chemotaxis has been shown to require both calcium and magnesium. Exposure of granulocytes to three different chemotactic factors (C5a, kallikrein, and dialyzable transfer factor) yielded a rapid calcium release, depressed calcium uptake, and was associated with a shift of calcium out of the cytoplasm and into a granule fraction. Colchicine, sodium azide, and cytochalasin B, in concentrations that inhibited chemotaxis, also inhibited calcium release while low concentrations of cytochalasin B, which enhanced chemotaxis, also enhanced calcium release. Microtubule assembly was visualized both in cells suspended in C5a without a chemotactic gradient and in cells actively migrating through a Micropore filter. The data suggest microtubule assembly is regulated, at least, in part, by the level of cytoplasmic calcium. It is proposed that asymmetric assembly of microtubules may be instrumental in imparting the net vector of motion during chemotaxis. PMID:4855032

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells.

PubMed

Melchior, Benoît; Frangos, John A

2012-08-15

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.

Magnolol and honokiol regulate the calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli-induced diarrhea mice.

PubMed

Deng, Yanli; Han, Xuefeng; Tang, Shaoxun; Xiao, Wenjun; Tan, Zhiliang; Zhou, Chuanshe; Wang, Min; Kang, Jinghe

2015-05-15

To explore the regulatory mechanisms of magnolol and honokiol on calcium-activated potassium channels signaling pathway in Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea mice, the concentrations of serum chloride ion (Cl(-)), sodium ion (Na(+)), potassium ion (K(+)) and calcium ion (Ca(2+)) were measured. Additionally, the mRNA expressions of calmodulin 1 (CaM), calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) and beta subunit (CaMKIIβ), ryanodine receptor 1, inositol 1,4,5-trisphosphate receptors (IP3 receptors), protein kinases C (PKC), potassium intermediate/small conductance calcium-activated channels (SK) and potassium large conductance calcium-activated channels(BK)were determined. A diarrhea mouse model was established using ETEC suspensions (3.29×10(9)CFU/ml) at a dosage of 0.02ml/g live body weight (BW). Magnolol or honokiol was intragastrically administered at dosages of 100 (M100 or H100), 300 (M300 or H300) and 500 (M500 or H500) mg/kg BW according to a 3×3 factorial arrangement. Magnolol and honokiol increased the Cl(-) and K(+) concentrations, further, upregulated the CaM, BKα1 and BKβ3 mRNA levels but downregulated the IP3 receptors 1, PKC, SK1, SK2, SK3, SK4 and BKβ4 mRNA expressions. Magnolol and honokiol did not alter the CaMKIIα, CaMKIIβ, ryanodine receptor 1, IP3 receptor 2, IP3 receptor 3, BKβ1 and BKβ2 mRNA expressions. These results clarify that magnolol and honokiol, acting through Ca(2+) channel blockade, inhibit the activation of IP3 receptor 1 to regulate the IP3-Ca(2+) store release, activate CaM to inhibit SK channels, and effectively suppress PKC kinases to promote BKα1 and BKβ3 channels opening and BKβ4 channel closing, which modulates the intestinal ion secretion. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Dynamics Simulations of Membrane-Bound STIM1 to Investigate Conformational Changes during STIM1 Activation upon Calcium Release.

PubMed

Mukherjee, Sreya; Karolak, Aleksandra; Debant, Marjolaine; Buscaglia, Paul; Renaudineau, Yves; Mignen, Olivier; Guida, Wayne C; Brooks, Wesley H

2017-02-27

Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformational changes accompanying homo- or heterodimerization of STIM1. For this purpose, the ER luminal portion of STIM1 (residues 58-236), which includes the sterile alpha motif (SAM) domain plus the calcium-binding EF-hand domains 1 and 2 attached to the STIM1 transmembrane region (TM), was modeled and embedded in a virtual membrane. Next, molecular dynamics simulations were performed to study the conformational changes that take place during STIM1 activation and subsequent protein-protein interactions. Indeed, the simulations revealed exposure of residues in the EF-hand domains, which may be important for dimerization steps. Altogether, understanding conformational changes in STIM1 can help in drug discovery when targeting this key protein in intracellular calcium functions.

A microstructural study of the degradation and calcium release from hydroxyapatite-calcium oxide ceramics made by infiltration.

PubMed

Zhang, Qinghao; Schmelzer, Eva; Gerlach, Jörg C; Nettleship, Ian

2017-04-01

Hydroxyapatite pellets, partially densified in a low-temperature heat treatment, were infiltrated with calcium nitrate solution followed by in-situ precipitation of Ca(OH) 2 and CaCO 3 . The infiltrated bodies were then densified to high relative density and the calcium carbonate transformed to calcium oxide during sintering and resulted in biphasic hydroxyapatite-CaO ceramics. This work investigated the influence of the infiltration on surface morphology, weight change, and microstructural-level degradation caused by exposure to saline at pH=7.4 and a temperature of 20°C. The CaO rendered the materials more susceptible to degradation, and released calcium into the saline faster than single phase, calcium deficient hydroxyapatite (HA) that were used as a control. In consequence, these ceramics could be used to release calcium into the culture microenvironments of bone tissue or bone marrow cells next to a scaffold surface. Copyright © 2016 Elsevier B.V. All rights reserved.

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

PubMed

Stern, Michael D; Cheng, Heping

2004-06-01

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.

Mitochondrial permeability transition in the crustacean Artemia franciscana: absence of a calcium-regulated pore in the face of profound calcium storage.

PubMed

Menze, Michael A; Hutchinson, Kirk; Laborde, Susan M; Hand, Steven C

2005-07-01

When mammalian mitochondria are exposed to high calcium and phosphate, a massive swelling, uncoupling of respiration, and release of cytochrome c occur. These changes are mediated by opening of the mitochondrial permeability transition pore (MPTP). Activation of the MPTP in vivo in response to hypoxic and oxidative stress leads to necrotic and apoptotic cell death. Considering that embryos of the brine shrimp Artemia franciscana tolerate anoxia for years, we investigated the MPTP in this crustacean to reveal whether pore opening occurs. Minimum molecular constituents of the regulated MPTP in mammals are believed to be the voltage-dependent anion channel, the adenine nucleotide translocators, and cyclophilin D. Western blot analysis revealed that mitochondria from A. franciscana possess all three required components. When measured with a calcium-sensitive fluorescent probe, rat liver mitochondria are shown to release matrix calcium after addition of >/=100 microM extramitochondrial calcium (MPTP opening), whereas brine shrimp mitochondria continue to take up extramitochondrial calcium and do not release internal stores even up to 1.0 mM exogenously added calcium (no MPTP opening). Furthermore, no swelling of A. franciscana mitochondria in response to added calcium was observed, and no release of cytochrome c could be detected. HgCl(2)-dependent swelling and cytochrome c release were readily confirmed, which is consistent with the presence of an "unregulated pore." Although the absence of a regulated MPTP in A. franciscana mitochondria could contribute to the extreme hypoxia tolerance in this species, we speculate that absence of the regulated MPTP may be a general feature of invertebrates.

The effect of radiopacifiers agents on pH, calcium release, radiopacity, and antimicrobial properties of different calcium hydroxide dressings.

PubMed

Ordinola-Zapata, Ronald; Bramante, Clovis Monteiro; García-Godoy, Franklin; Moldauer, Bertram Ivan; Gagliardi Minotti, Paloma; Tercília Grizzo, Larissa; Duarte, Marco Antonio Hungaro

2015-07-01

The aim of this study was to evaluate the antimicrobial activity, pH level, calcium ion release, and radiopacity of calcium hydroxide pastes associated with three radiopacifying agents (iodoform, zinc oxide, and barium sulfate). For the pH and calcium release tests, 45 acrylic teeth were utilized and immersed in ultrapure water. After 24 h, 72 h, and 7 days the solution was analyzed by using a pH meter and an atomic absorption spectrophotometer. Polyethylene tubes filled with the pastes were used to perform the radiopacity test. For the antimicrobial test, 25 dentin specimens were infected intraorally in order to induce the biofilm colonization and treated with the pastes for 7 days. The Live/Dead technique and a confocal microscope were used to obtain the ratio of live cells. Parametric and nonparametric statistical tests were performed to show differences among the groups (P < 0.05). The pH analysis at 7 days showed significant differences (P < 0.05) among the groups. No differences among the pastes were found in the calcium release test on the 7th day (P > 0.05). The calcium hydroxide/iodoform samples had the highest radiopacity and antimicrobial activity against the biofilm-infected dentin in comparison to the other pastes (P < 0.05). Calcium hydroxide mixed with 17% iodoform and 35% propylene glycol into a paste had the highest pH, calcium ion release, radiopacity, and the greatest antimicrobial action versus similar samples mixed with BaSO4 or ZnO. © 2015 Wiley Periodicals, Inc.

Effects of calcium hydroxide addition on the physical and chemical properties of a calcium silicate-based sealer

PubMed Central

KUGA, Milton Carlos; DUARTE, Marco Antonio Hungaro; SANT'ANNA-JÚNIOR, Arnaldo; KEINE, Kátia Cristina; FARIA, Gisele; DANTAS, Andrea Abi Rached; GUIOTTI, Flávia Angélica

2014-01-01

Recently, various calcium silicate-based sealers have been introduced for use in root canal filling. The MTA Fillapex is one of these sealers, but some of its physicochemical properties are not in accordance with the ISO requirements. Objective The aim of this study was to evaluate the flowability, pH level and calcium release of pure MTA Fillapex (MTAF) or containing 5% (MTAF5) or 10% (MTAF10) calcium hydroxide (CH), in weight, in comparison with AH Plus sealer. Material and Methods The flowability test was performed according to the ISO 6876:2001 requirements. For the pH level and calcium ion release analyses, the sealers were placed individually (n=10) in plastic tubes and immersed in deionized water. After 24 hours, 7 and 14 days, the water in which each specimen had been immersed was evaluated to determine the pH level changes and calcium released. Flowability, pH level and calcium release data were analyzed statistically by the ANOVA test (α=5%). Results In relation to flowability: MTAF>AH Plus>MTAF5>MTAF10. In relation to the pH level, for 24 h: MTAF5=MTAF10=MTAF>AH Plus; for 7 and 14 days: MTAF5=MTAF10>MTAF>AH Plus. For the calcium release, for all periods: MTAF>MTAF5=MTAF10>AH Plus. Conclusions The addition of 5% CH to the MTA Fillapex (in weight) is an alternative to reduce the high flowability presented by the sealer, without interfering in its alkalization potential. PMID:25025558

PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

PubMed Central

Chen, Ruichuan; Liu, Min; Li, Huan; Xue, Yuhua; Ramey, Wanichaya N.; He, Nanhai; Ai, Nanping; Luo, Haohong; Zhu, Ying; Zhou, Nan; Zhou, Qiang

2008-01-01

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca2+)–calmodulin–protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1α (PP1α) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1α releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1α alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription. PMID:18483222

Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity*

PubMed Central

Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T.

2011-01-01

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. PMID:21296883

Calcium-binding proteins and development

NASA Technical Reports Server (NTRS)

Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

1998-01-01

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

Calcium-Induced Calcium Release during Action Potential Firing in Developing Inner Hair Cells

PubMed Central

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

2015-01-01

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 into the calcium signaling mechanisms involved in early developmental processes. PMID:25762313

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

PubMed

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 into the calcium signaling mechanisms involved in early developmental processes. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Growth differentiation factor-15 promotes glutamate release in medial prefrontal cortex of mice through upregulation of T-type calcium channels.

PubMed

Liu, Dong-Dong; Lu, Jun-Mei; Zhao, Qian-Ru; Hu, Changlong; Mei, Yan-Ai

2016-06-29

Growth differentiation factor-15 (GDF-15) has been implicated in ischemic brain injury and synapse development, but its involvement in modulating neuronal excitability and synaptic transmission remain poorly understood. In this study, we investigated the effects of GDF-15 on non-evoked miniature excitatory post-synaptic currents (mEPSCs) and neurotransmitter release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with GDF-15 for 60 min significantly increased the frequency of mEPSCs without effect on their amplitude. GDF-15 also significantly elevated presynaptic glutamate release, as shown by HPLC. These effects were blocked by dual TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII) antagonists, but not by a TβRI antagonist alone. Meanwhile, GDF-15 enhanced pERK level, and inhibition of MAPK/ERK activity attenuated the GDF-15-induced increases in mEPSC and glutamate release. Blocking T-type calcium channels reduced the GDF-15 induced up-regulation of synaptic transmission. Membrane-protein extraction and use of an intracellular protein-transport inhibitor showed that GDF-15 promoted CaV3.1 and CaV3.3 α-subunit expression by trafficking to the membrane. These results confirm previous findings in cerebellar granule neurons, in which GDF-15 induces its neurobiological effects via TβRII and activation of the ERK pathway, providing novel insights into the mechanism of GDF-15 function in cortical neurons.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells.

PubMed

Chang, John P; Sawisky, Grant R; Davis, Philip J; Pemberton, Joshua G; Rieger, Aja M; Barreda, Daniel R

2014-09-15

Nitric oxide (NO) and Ca(2+) are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca(2+) signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca(2+)-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca(2+) availability and voltage-sensitive Ca(2+) channels, as well as intracellular Ca(2+) store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca(2+)-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca(2+)-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca(2+)-dependent transduction pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential calcium dependence in basal and forskolin-potentiated spontaneous transmitter release in basolateral amygdala neurons.

PubMed

Miura, Yuki; Naka, Masamitsu; Matsuki, Norio; Nomura, Hiroshi

2012-10-31

Action potential-independent transmitter release, or spontaneous release, is postulated to produce multiple postsynaptic effects (e.g., maintenance of dendritic spines and suppression of local dendritic protein synthesis). Potentiation of spontaneous release may contribute to the precise modulation of synaptic function. However, the expression mechanism underlying potentiated spontaneous release remains unclear. In this study, we investigated the involvement of extracellular and intracellular calcium in basal and potentiated spontaneous release. Miniature excitatory postsynaptic currents (mEPSCs) of the basolateral amygdala neurons in acute brain slices were recorded. Forskolin, an adenylate cyclase activator, increased mEPSC frequency, and the increase lasted at least 25 min after washout. Removal of the extracellular calcium decreased mEPSC frequency in both naïve and forskolin-treated slices. On the other hand, chelation of intracellular calcium by BAPTA-AM decreased mEPSC frequency in naïve, but not in forskolin-treated slices. A blockade of the calcium-sensing receptor (CaSR) resulted in an increase in mEPSC frequency in forskolin-treated, but not in naïve slices. These findings indicate that forskolin-induced potentiation is accompanied by changes in the mechanisms underlying Ca(2+)-dependent spontaneous release. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

PubMed Central

Mandal, Amritlal; Delamere, Nicholas A.

2010-01-01

Purpose. Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90RSK and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation. Methods. Cytoplasmic calcium concentration ([Ca2+]i) was measured in cultured rat optic nerve astrocytes loaded with fura-2. The cells were placed in a closed imaging chamber and subjected to an HP increase of 15 mm Hg. Protein phosphorylation was detected by Western blot analysis. Results. The increase of HP caused an immediate slow increase in [Ca2+]i. The response persisted in calcium-free solution and when nickel chloride (4 mM) was added to suppress channel-mediated calcium entry. Previous depletion of the ER calcium stores by cyclopiazonic acid abolished the HP-induced calcium level increase. The HP-induced increase persisted in cells exposed to xestospongin C, an inhibitor of IP3R-mediated calcium release. In contrast, ryanodine receptor (RyR) antagonist ruthenium red (10 μM) or dantrolene (25 μM) inhibited the HP-induced calcium increase. The HP-induced calcium increase was abolished when ryanodine-sensitive calcium stores were pre-depleted with caffeine (3 mM). HP caused ERK1/2 phosphorylation. The magnitude of the ERK1/2 phosphorylation response was reduced by ruthenium red and dantrolene. Conclusions. Increasing HP causes calcium release from a ryanodine-sensitive cytoplasmic store and subsequent ERK1/2 activation. Calcium store release appears to be a required early step in the initial astrocyte response to an HP increase. PMID:20071675

Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism

PubMed Central

Petrou, Terry; Olsen, Hervør L.; Thrasivoulou, Christopher; Masters, John R.; Ashmore, Jonathan F.

2017-01-01

Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i. We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds. PMID:27980039

Inositol trisphosphate receptor mediated spatiotemporal calcium signalling.

PubMed

Miyazaki, S

1995-04-01

Spatiotemporal Ca2+ signalling in the cytoplasm is currently understood as an excitation phenomenon by analogy with electrical excitation in the plasma membrane. In many cell types, Ca2+ waves and Ca2+ oscillations are mediated by inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channels in the endoplasmic reticulum membrane, with positive feedback between cytosolic Ca2+ and IP3-induced Ca2+ release creating a regenerative process. Remarkable advances have been made in the past year in the analysis of subcellular Ca2+ microdomains using confocal microscopy and of Ca2+ influx pathways that are functionally coupled to IP3-induced Ca2+ release. Ca2+ signals can be conveyed into the nucleus and mitochondria. Ca2+ entry from outside the cell allows repetitive Ca2+ release by providing Ca2+ to refill the endoplasmic reticulum stores, thus giving rise to frequency-encoded Ca2+ signals.

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle.

PubMed

Szentesi, Péter; Szappanos, Henrietta; Szegedi, Csaba; Gönczi, Monika; Jona, István; Cseri, Julianna; Kovács, László; Csernoch, László

2004-03-01

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

PubMed Central

Lu, Ben; Kwan, Kevin; Levine, Yaakov A; Olofsson, Peder S; Yang, Huan; Li, Jianhua; Joshi, Sonia; Wang, Haichao; Andersson, Ulf; Chavan, Sangeeta S; Tracey, Kevin J

2014-01-01

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release. PMID:24849809

Fluoride-containing nanoporous calcium-silicate MTA cements for endodontics and oral surgery: early fluorapatite formation in a phosphate-containing solution.

PubMed

Gandolfi, M G; Taddei, P; Siboni, F; Modena, E; Ginebra, M P; Prati, C

2011-10-01

To test the chemical-physical properties and apatite-forming ability of experimental fluoride-doped calcium silicate cements designed to create novel bioactive materials for use in endodontics and oral surgery. A thermally treated calcium silicate cement (wTC) containing CaCl(2) 5%wt was modified by adding NaF 1%wt (FTC) or 10%wt (F10TC). Cements were analysed by environmental scanning electron microscopy with energy-dispersive X-ray analysis, IR and micro-Raman spectroscopy in wet conditions immediately after preparation or after ageing in a phosphate-containing solution (Dulbecco's phosphate-buffered saline). Calcium and fluoride release and pH of the storage solution were measured. The results obtained were analysed statistically (Tukey's HSD test and two-way anova). The formation of calcium phosphate precipitates (spherulites) was observed on the surface of 24 h-aged cements and the formation of a thick bone-like B-type carbonated apatite layer (biocoating) on 28 day-aged cements. The rate of apatite formation was FTC>F10TC>wTC. Fluorapatite was detected on FTC and F10TC after 1 day of ageing, with a higher fluoride content on F10TC. All the cements released calcium ions. At 5 and 24 h, the wTC had the significantly highest calcium release (P<0.001) that decreased significantly over the storage time. At 3-28 days, FTC and F10TC had significantly higher calcium release than wTC (P<0.05). The F10TC had the significantly highest fluoride release at all times (P<0.01) that decreased significantly over storage time. No significant differences were observed between FTC and wTC. All the cements had a strong alkalinizing activity (OH(-) release) that remained after 28 days of storage. The addition of sodium fluoride accelerated apatite formation on calcium silicate cements. Fluoride-doped calcium silicate cements had higher bioactivity and earlier formation of fluorapatite. Sodium fluoride may be introduced in the formulation of mineral trioxide aggregate cements to enhance their biological behaviour. F-doped calcium silicate cements are promising bone cements for clinical endodontic use. © 2011 International Endodontic Journal.

Transcriptional Regulation of Mouse Ribosomal RNA Synthesis by Pathways Involving Protein Kinase C, Calcium, Insulin and Serum

DTIC Science & Technology

1992-05-08

Southern California 1ha.iITRINUTIONI AVAILASILITY STATEMENT 12b. DITIUINCCD1 This document has been approved { OSRUTO for public release and sale ...and 500 gIl of CHCI3 : Isoamyl alcohol (24 : 1), and 2X with 1ml of CHCI 3 : Iso amyl alcohol (24 : 1). RNA was precipitated at -200C overnight in the...control. Plasmid constructions. Plasmids pMrA and pMrD (Kuhn and Grummt, 1987) were kind gifts of Dr. Ingrid Grummt. Plasmid p119 and p123 (Arnhiem

pH and calcium ion release evaluation of pure and calcium hydroxide-containing Epiphany for use in retrograde filling

PubMed Central

TANOMARU-FILHO, Mário; SAÇAKI, Juliana Nogueira; FALEIROS, Frederico Bordini Chaves; GUERREIRO-TANOMARU, Juliane Maria

2011-01-01

Objective Hydroxyl (OH-) and calcium (Ca++) ion release was evaluated in six materials: G1) Sealer 26, G2) White mineral trioxide aggregate (MTA), G3) epiphany, G4) epiphany + 10% calcium hydroxide (CH), G5) epiphany + 20% CH, and G6) zinc oxide and eugenol. Material and Methods Specimens were placed in polyethylene tubes and immersed in distilled water. After 3, 6, 12, 24, and 48 h, 7, 14, and 28 days, the water was assessed for pH with a pH meter and for Ca++ release by atomic absorption spectrophotometry. Results G1, G2, G4, and G5 had the highest pH until 14 days (p<0.05). G1 presented the highest Ca++ release until 6 h, and G4 and G5, from 12 h through 14 days. Ca++ release was greater for G1 and G2 at 28 days. G6 released the least Ca++. Conclusion MTA, Sealer 26, epiphany, and epiphany + CH release OH - and Ca++ ions. Epiphany + CH may be an alternative as retrofilling material. PMID:21437461

A Molecular Web: Endoplasmic Reticulum Stress, Inflammation, and Oxidative Stress

PubMed Central

Chaudhari, Namrata; Talwar, Priti; Parimisetty, Avinash; Lefebvre d’Hellencourt, Christian; Ravanan, Palaniyandi

2014-01-01

Execution of fundamental cellular functions demands regulated protein folding homeostasis. Endoplasmic reticulum (ER) is an active organelle existing to implement this function by folding and modifying secretory and membrane proteins. Loss of protein folding homeostasis is central to various diseases and budding evidences suggest ER stress as being a major contributor in the development or pathology of a diseased state besides other cellular stresses. The trigger for diseases may be diverse but, inflammation and/or ER stress may be basic mechanisms increasing the severity or complicating the condition of the disease. Chronic ER stress and activation of the unfolded-protein response (UPR) through endogenous or exogenous insults may result in impaired calcium and redox homeostasis, oxidative stress via protein overload thereby also influencing vital mitochondrial functions. Calcium released from the ER augments the production of mitochondrial Reactive Oxygen Species (ROS). Toxic accumulation of ROS within ER and mitochondria disturbs fundamental organelle functions. Sustained ER stress is known to potentially elicit inflammatory responses via UPR pathways. Additionally, ROS generated through inflammation or mitochondrial dysfunction could accelerate ER malfunction. Dysfunctional UPR pathways have been associated with a wide range of diseases including several neurodegenerative diseases, stroke, metabolic disorders, cancer, inflammatory disease, diabetes mellitus, cardiovascular disease, and others. In this review, we have discussed the UPR signaling pathways, and networking between ER stress-induced inflammatory pathways, oxidative stress, and mitochondrial signaling events, which further induce or exacerbate ER stress. PMID:25120434

A proteomics study of hyperhomocysteinemia injury of the hippocampal neurons using iTRAQ.

PubMed

Fang, Min; Wang, Jing; Yan, Han; Zhao, Yan-Xin; Liu, Xue-Yuan

2014-11-01

High levels of homocysteine, caused by abnormal methionine metabolism, can induce degeneration of mouse hippocampal neurons. iTRAQ™ technology has been widely used in the field of proteomics research and through employing this technology, the present study identified that hyperhomocysteinemia induced the downregulation of 52 proteins and upregulation of 44 proteins in the mouse hippocampus. Through gene ontology and pathway analysis, the upregulation of components of the cytoskeleton, actin, regulators of focal adhesion, calcium signaling pathways, tight junctions, ErbB and gonadotrophin‑releasing hormone signaling, leukocyte, transendothelial migration, propanoate and pyruvate metabolism, valine, leucine and isoleucine biosynthesis, synthesis and degradation of ketone bodies and benzoate degradation via CoA ligation pathway, was identified. It was additionally verified that tau protein was highly expressed in the hyperhomocysteinemic neurons. Further analysis revealed that tau network proteins played functional roles in homocysteine‑induced neuronal damage.

Fast Kinetics of Calcium Signaling and Sensor Design

PubMed Central

Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

2015-01-01

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway

PubMed Central

Haustein, Martin D.; Kracun, Sebastian; Lu, Xiao-Hong; Shih, Tiffany; Jackson-Weaver, Olan; Tong, Xiaoping; Xu, Ji; Yang, X. William; O'Dell, Thomas J.; Marvin, Jonathan S.; Ellisman, Mark H.; Bushong, Eric A.; Looger, Loren L.; Khakh, Baljit S.

2014-01-01

Summary The spatiotemporal activities of astrocyte Ca2+ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca2+ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localised spontaneous Ca2+ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca2+ signaling changed linearly with the number of mossy fiber action potentials. Under these settings astrocyte responses were global, suppressed by neurotransmitter clearance and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca2+ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway. PMID:24742463

PYK2: A Calcium-sensitive Protein Tyrosine Kinase Activated in Response to Fertilization of the Zebrafish Oocyte

PubMed Central

Sharma, Dipika; Kinsey, William H.

2012-01-01

Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells.

PubMed

Liu, Lei-Lei; Deng, Qin-Qin; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

2016-09-22

Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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.

Properties of a novel polysiloxane-guttapercha calcium silicate-bioglass-containing root canal sealer.

PubMed

Gandolfi, M G; Siboni, F; Prati, C

2016-05-01

Root canal filling sealers based on polymethyl hydrogensiloxane or polymethyl hydrogensiloxane-guttapercha--introduced to improve the quality of conventional guttapercha-based and resin-based systems--showed advantages in handiness and clinical application. The aim of the study was to evaluate the chemical-physical properties of a novel polysiloxane-guttapercha calcium silicate-containing root canal sealer (GuttaFlow bioseal). GuttaFlow bioseal was examined and compared with GuttaFlow2, RoekoSeal and MTA Fillapex sealers. Setting times, open and impervious porosity and apparent porosity, water sorption, weight loss, calcium release, and alkalinizing activity were evaluated. ESEM-EDX-Raman analyses of fresh materials and after soaking in simulated body fluid were also performed. Marked differences were obtained among the materials. GuttaFlow bioseal showed low solubility and porosity, high water sorption, moderate calcium release and good alkalinizing activity. MTA Fillapex showed the highest calcium release, alkalinizing activity and solubility, RoekoSeal the lowest calcium release, no alkalinizing activity, very low solubility and water sorption. Only GuttaFlow bioseal showed apatite forming ability. GuttaFlow bioseal showed alkalinizing activity together with negligible solubility and slight calcium release. Therefore, the notable nucleation of apatite and apatite precursors can be related to the co-operation of CaSi particles (SiOH groups) with polysiloxane (SiOSi groups). The incorporation of a calcium silicate component into polydimethyl polymethylhydrogensiloxane guttapercha sealers may represent an attractive strategy to obtain a bioactive biointeractive flowable guttapercha sealer for moist/bleeding apices with bone defects in endodontic therapy. Copyright © 2016 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

PubMed

Stefano, G B; Prevot, V; Beauvillain, J C; Fimiani, C; Welters, I; Cadet, P; Breton, C; Pestel, J; Salzet, M; Bilfinger, T V

1999-10-01

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

Studying dyadic structure-function relationships: a review of current modeling approaches and new insights into Ca2+ (mis)handling.

PubMed

Maleckar, Mary M; Edwards, Andrew G; Louch, William E; Lines, Glenn T

2017-01-01

Excitation-contraction coupling in cardiac myocytes requires calcium influx through L-type calcium channels in the sarcolemma, which gates calcium release through sarcoplasmic reticulum ryanodine receptors in a process known as calcium-induced calcium release, producing a myoplasmic calcium transient and enabling cardiomyocyte contraction. The spatio-temporal dynamics of calcium release, buffering, and reuptake into the sarcoplasmic reticulum play a central role in excitation-contraction coupling in both normal and diseased cardiac myocytes. However, further quantitative understanding of these cells' calcium machinery and the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease requires accurate knowledge of cardiac ultrastructure, protein distribution and subcellular function. As current imaging techniques are limited in spatial resolution, limiting insight into changes in calcium handling, computational models of excitation-contraction coupling have been increasingly employed to probe these structure-function relationships. This review will focus on the development of structural models of cardiac calcium dynamics at the subcellular level, orienting the reader broadly towards the development of models of subcellular calcium handling in cardiomyocytes. Specific focus will be given to progress in recent years in terms of multi-scale modeling employing resolved spatial models of subcellular calcium machinery. A review of the state-of-the-art will be followed by a review of emergent insights into calcium-dependent etiologies in heart disease and, finally, we will offer a perspective on future directions for related computational modeling and simulation efforts.

Genetic analysis of hyperemesis gravidarum reveals association with intracellular calcium release channel (RYR2).

PubMed

Fejzo, Marlena Schoenberg; Myhre, Ronny; Colodro-Conde, Lucía; MacGibbon, Kimber W; Sinsheimer, Janet S; Reddy, M V Prasad Linga; Pajukanta, Päivi; Nyholt, Dale R; Wright, Margaret J; Martin, Nicholas G; Engel, Stephanie M; Medland, Sarah E; Magnus, Per; Mullin, Patrick M

2017-01-05

Hyperemesis Gravidarum (HG), severe nausea/vomiting in pregnancy (NVP), can cause poor maternal/fetal outcomes. Genetic predisposition suggests the genetic component is essential in discovering an etiology. We performed whole-exome sequencing of 5 families followed by analysis of variants in 584 cases/431 controls. Variants in RYR2 segregated with disease in 2 families. The novel variant L3277R was not found in any case/control. The rare variant, G1886S was more common in cases (p = 0.046) and extreme cases (p = 0.023). Replication of G1886S using Norwegian/Australian data was supportive. Common variants rs790899 and rs1891246 were significantly associated with HG and weight loss. Copy-number analysis revealed a deletion in a patient. RYR2 encodes an intracellular calcium release channel involved in vomiting, cyclic-vomiting syndrome, and is a thyroid hormone target gene. Additionally, RYR2 is a downstream drug target of Inderal, used to treat HG and CVS. Thus, herein we provide genetic evidence for a pathway and therapy for HG. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

PubMed

Gobin, V; De Bock, M; Broeckx, B J G; Kiselinova, M; De Spiegelaere, W; Vandekerckhove, L; Van Steendam, K; Leybaert, L; Deforce, D

2015-09-01

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Properties of calcium silicate-monobasic calcium phosphate materials for endodontics containing tantalum pentoxide and zirconium oxide.

PubMed

Zamparini, Fausto; Siboni, Francesco; Prati, Carlo; Taddei, Paola; Gandolfi, Maria Giovanna

2018-05-08

The aim of the study was to evaluate chemical-physical properties and apatite-forming ability of three premixed calcium silicate materials containing monobasic calcium phosphate (CaH 4 P 2 O 8 ) bioceramic, tantalum pentoxide and zirconium oxide, recently marketed for endodontics (TotalFill BC-Sealer, BC-RRM-Paste, BC-RRM-Putty). Microchemical and micromorphological analyses, radiopacity, initial and final setting times, calcium release and alkalising activity were tested. The nucleation of calcium phosphates (CaPs) and/or apatite after 28 days ageing was evaluated by ESEM-EDX and micro-Raman spectroscopy. BC-Sealer and BC-RRM-Paste showed similar initial (23 h), prolonged final (52 h) setting times and good radiopacity (> 7 mm Al); BC-RRM-Putty showed fast initial (2 h) and final setting times (27 h) and excellent radiopacity (> 9 mm Al). All materials induced a marked alkalisation (pH 11-12) up to 28 days and showed the release of calcium ions throughout the entire test period (cumulative calcium release 641-806 ppm). After 28 days ageing, a well-distributed mineral layer was present on all samples surface; EDX demonstrated relevant calcium and phosphorous peaks. B-type carbonated apatite and calcite deposits were identified by micro-Raman spectroscopy on all the 28-day-aged samples; the deposit thickness was higher on BC-RRM-Paste and BC-RRM-Putty, in agreement with calcium release data. These materials met the required chemical and physical standards and released biologically relevant ions. The CaSi-CaH 4 P 2 O 8 system present in the materials provided Ca and OH ions release with marked abilities to nucleate a layer of B-type carbonated apatite favoured/accelerated by the bioceramic presence. The ability to nucleate apatite may lead many clinical advantages: In orthograde endodontics, it may improve the sealing ability by the deposition of CaPs at the material-root dentine interface, and in endodontic surgery, it could promote bone and periodontal tissue regeneration. As premixed materials, their application in endodontics may result easier in several complex endodontic situations (apicoectomy, root perforation, presence of wide/wet apices).

Co-occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess.

PubMed

Gonzalez, Alberto; Vera, Jeannette; Castro, Jorge; Dennett, Geraldine; Mellado, Macarena; Morales, Bernardo; Correa, Juan A; Moenne, Alejandra

2010-10-01

In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes. © 2010 Blackwell Publishing Ltd.

Effects of inorganic phosphate and ADP on calcium handling by the sarcoplasmic reticulum in rat skinned cardiac muscles.

PubMed

Xiang, J Z; Kentish, J C

1995-03-01

The aim was to investigate whether, and how, increases in inorganic phosphate (Pi) and ADP, similar to those occurring intracellularly during early myocardial ischaemia, affect the calcium handling of the sarcoplasmic reticulum. Rat ventricular trabeculae were permeabilised with saponin. The physiological process of calcium induced calcium release (CICR) from the muscle sarcoplasmic reticulum was triggered via flash photolysis of the "caged Ca2+", nitr-5. Alternatively, calcium release was induced by rapid application of caffeine to give an estimate of sarcoplasmic reticular calcium loading. The initial rate of sarcoplasmic reticular calcium pumping was also assessed by photolysis of caged ATP at saturating [Ca2+]. Myoplasmic [Ca2+] (using fluo-3) and isometric force were measured. Pi (2-20 mM) significantly depressed the magnitude of CICR and the associated force transient. Sarcoplasmic reticular calcium loading was inhibited even more than CICR by Pi, suggesting that reduced calcium loading could account for all of the inhibitory effect of Pi on CICR and that Pi may slightly activate the calcium release mechanism. The reduced sarcoplasmic reticular calcium loading seemed to be due to a fall in the free energy of ATP hydrolysis (delta GATP) available for the calcium pump, since equal decreases in delta GATP produced by adding both Pi and ADP in various ratios caused similar falls in the calcium loading of the sarcoplasmic reticulum. The caged ATP experiments indicated that Pi (20 mM) did not affect the rate constant of sarcoplasmic reticular calcium uptake. ADP (10 mM) alone, or with 1 mM Pi, inhibited calcium loading. In spite of this, ADP (10 mM) did not alter CICR and, when 1 mM Pi was added, ADP increased CICR above control. An increase in intracellular Pi reduces sarcoplasmic reticular calcium loading and thus depresses the CICR. This could be an important contributing factor in the hypoxic or ischaemic contractile failure of the myocardium. However the detrimental effect of Pi may be offset to some extent by a stimulatory action of ADP on the calcium release mechanism of CICR.

Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

PubMed

Orellana, D I; Quintanilla, R A; Gonzalez-Billault, C; Maccioni, R B

2005-11-01

Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns.

Contributions of two types of calcium channels to synaptic transmission and plasticity.

PubMed

Edmonds, B; Klein, M; Dale, N; Kandel, E R

1990-11-23

In Aplysia sensory and motor neurons in culture, the contributions of the major classes of calcium current can be selectively examined while transmitter release and its modulation are examined. A slowly inactivating, dihydropyridine-sensitive calcium current does not contribute either to normal synaptic transmission or to any of three different forms of plasticity: presynaptic inhibition, homosynaptic depression, and presynaptic facilitation. This current does contribute, however, to a fourth form of plasticity--modulation of transmitter release by tonic depolarization of the sensory neuron. By contrast, a second calcium current, which is rapidly inactivating and dihydropyridine-insensitive, contributes to release elicited by the transient depolarization of an action potential and to the other three forms of plasticity.

Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

PubMed Central

Nebl, Thomas; Prieto, Judith Helena; Kapp, Eugene; Smith, Brian J.; Williams, Melanie J.; Yates, John R.; Cowman, Alan F.; Tonkin, Christopher J.

2011-01-01

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. PMID:21980283

Readily releasable pool of synaptic vesicles measured at single synaptic contacts.

PubMed

Trigo, Federico F; Sakaba, Takeshi; Ogden, David; Marty, Alain

2012-10-30

To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

Membrane properties involved in calcium-stimulated microparticle release from the plasma membranes of S49 lymphoma cells.

PubMed

Campbell, Lauryl E; Nelson, Jennifer; Gibbons, Elizabeth; Judd, Allan M; Bell, John D

2014-01-01

This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32-42°C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes.

Platelet-derived Growth Factor-mediated Induction of the Synaptic Plasticity Gene Arc/Arg3.1*

PubMed Central

Peng, Fuwang; Yao, Honghong; Bai, Xuetao; Zhu, Xuhui; Reiner, Benjamin C.; Beazely, Michael; Funa, Keiko; Xiong, Huangui; Buch, Shilpa

2010-01-01

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc. PMID:20452974

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

PubMed

King, Justin R; Ullah, Aman; Bak, Ellen; Jafri, M Saleet; Kabbani, Nadine

2018-06-01

The pharmacological targeting of the α 7 nicotinic acetylcholine receptor ( α 7) is a promising strategy in the development of new drugs for neurologic diseases. Because α 7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α 7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α 7 receptor. Both influx of calcium through the α 7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α 7 D44A or α 7 345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α 7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α 7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α 7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α 7 receptor allosteric modulation on both local and global calcium dynamics. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Calcium-independent haemolysis via the lectin pathway of complement activation in the guinea-pig and other *

PubMed Central

Zhang, Y; Suankratay, C; Zhang, X-H; Jones, D R; Lint, T F; Gewurz, H

1999-01-01

We previously reported that complement-dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan-binding lectin (MBL) via the lectin pathway in man occurs in Mg-EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E-M, but once the E-M-MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E-M-MBL in guinea-pig, rat, dog and pig sera, and was further investigated in the guinea-pig, in which titres were much higher (∼14-fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4- or C2-deficient guinea-pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea-pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G-75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea-pig serum to mediate haemolysis via the lectin pathway. Further, the C3-convertase formed via the lectin pathway (E-M-MBL-C4,2) lysed in C2-deficient guinea-pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea-pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea-pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology. PMID:10457224

Effect of sepsis on calcium uptake and content in skeletal muscle and regulation in vitro by calcium of total and myofibrillar protein breakdown in control and septic muscle: Results from a preliminary study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benson, D.W.; Hasselgren, P.O.; Hiyama, D.T.

Because high calcium concentration in vitro stimulates muscle proteolysis, calcium has been implicated in the pathogenesis of increased muscle breakdown in different catabolic conditions. Protein breakdown in skeletal muscle is increased during sepsis, but the effect of sepsis on muscle calcium uptake and content is not known. In this study the influence of sepsis, induced in rats by cecal ligation and puncture, on muscle calcium uptake and content was studied. Sixteen hours after cecal ligation and puncture or sham operation, calcium content of the extensor digitorum longus (EDL) and soleus (SOL) muscles was determined with an atomic absorption spectrometer. Calciummore » uptake was measured in intact SOL muscles incubated in the presence of calcium 45 (45Ca) for between 1 and 120 minutes. Total and myofibrillar protein breakdown was determined in SOL muscles, incubated in the presence of different calcium concentrations (0; 2.5; 5.0 mmol/L), and measured as release into the incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Calcium content was increased by 51% (p less than 0.001) during sepsis in SOL and by 10% (p less than 0.05) in EDL muscle. There was no difference in 45Ca uptake between control and septic muscles during the early phase (1 to 5 minutes) of incubation. During more extended incubation (30 to 120 minutes), muscles from septic rats took up significantly more 45Ca than control muscles (p less than 0.05). Tyrosine release by incubated SOL muscles from control and septic rats was increased when calcium was added to the incubation medium, and at a calcium concentration of 2.5 mmol/L, the increase in tyrosine release was greater in septic than in control muscle. Addition of calcium to the incubation medium did not affect 3-MH release in control or septic muscle.« less

Modification of caffeine-induced injury in Ca2+-free perfused rat hearts. Relationship to the calcium paradox.

PubMed Central

Vander Heide, R. S.; Altschuld, R. A.; Lamka, K. G.; Ganote, C. E.

1986-01-01

The pathogenesis of the calcium paradox has not been established. In calcium-free perfused hearts, caffeine, which releases calcium from the sarcoplasmic reticulum, causes severe myocardial injury, with creatine kinase (CK) release and contraction band necrosis similar in many respects to the calcium paradox. It has been postulated that contracture, initiated by a small rise in intracellular calcium, may cause sarcolemmal injury in both the calcium paradox and caffeine-induced myocardial injury. The present study was initiated to determine whether interventions which modulate caffeine-induced contracture will also correspondingly alter cellular injury. The effects of caffeine dose, procaine, extended calcium-free perfusion, elevated potassium, temperature, and increasing intracellular sodium on caffeine-induced contracture were examined in Langendorff-perfused adult rat hearts. Caffeine-induced contracture at 22 C increased over a dose range of 5-40 mM caffeine. Procaine, which inhibits caffeine-induced calcium release at doses between 5 and 20 mM, progressively reduced contracture caused by addition of 20 mM caffeine at 22 C. Hearts perfused with calcium-free solution containing 16 mM K+ showed a reduction in caffeine-induced contracture. Extended calcium-free perfusion (20 minutes) at temperatures from 18 to 37 C resulted in a progressive reduction of caffeine-induced contracture. Each of these interventions was also found to inhibit caffeine-induced injury at 37 C. Low temperature was found to have complex effects. Hypothermia enhanced caffeine contractures but also protected hearts from cell separations and CK release. Increasing intracellular sodium was found to enhance caffeine-induced contracture at 37 C. There was a direct correlation between measured intracellular sodium levels and the magnitude and duration of caffeine-induced contracture. These results demonstrate a direct correlation between the magnitude of contracture and myocardial injury in calcium-free hearts. It is proposed that contracture is the primary mediator of sarcolemmal membrane injury in hearts with intercalated disks weakened by prior calcium-free perfusion. Images Figure 11 PMID:3706496

Alternative Mechanism by which IFN-γ Enhances Tumor Recognition: Active Release of Heat Shock Protein 721

PubMed Central

Bausero, Maria A.; Gastpar, Robert; Multhoff, Gabriele; Asea, Alexzander

2006-01-01

IFN-γ exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-γ enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-γ triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-γ-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl β-cyclodextrin completely abrogated IFN-γ-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-γ promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal. PMID:16116176

Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72.

PubMed

Bausero, Maria A; Gastpar, Robert; Multhoff, Gabriele; Asea, Alexzander

2005-09-01

IFN-gamma exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-gamma enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-gamma triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-gamma-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl beta-cyclodextrin completely abrogated IFN-gamma-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-gamma promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.

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

PubMed

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.

Mitochondrial modulation-induced activation of vagal sensory neuronal subsets by antimycin A, but not CCCP or rotenone, correlates with mitochondrial superoxide production.

PubMed

Stanford, Katherine R; Taylor-Clark, Thomas E

2018-01-01

Inflammation causes nociceptive sensory neuron activation, evoking debilitating symptoms and reflexes. Inflammatory signaling pathways are capable of modulating mitochondrial function, resulting in reactive oxygen species (ROS) production, mitochondrial depolarization and calcium release. Previously we showed that mitochondrial modulation with antimycin A, a complex III inhibitor, selectively stimulated nociceptive bronchopulmonary C-fibers via the activation of transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) cation channels. TRPA1 is ROS-sensitive, but there is little evidence that TRPV1 is activated by ROS. Here, we used dual imaging of dissociated vagal neurons to investigate the correlation of mitochondrial superoxide production (mitoSOX) or mitochondrial depolarization (JC-1) with cytosolic calcium (Fura-2AM), following mitochondrial modulation by antimycin A, rotenone (complex I inhibitor) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent). Mitochondrial modulation by all agents selectively increased cytosolic calcium in a subset of TRPA1/TRPV1-expressing (A1/V1+) neurons. There was a significant correlation between antimycin A-induced calcium responses and mitochondrial superoxide in wild-type 'responding' A1/V1+ neurons, which was eliminated in TRPA1-/- neurons, but not TRPV1-/- neurons. Nevertheless, antimycin A-induced superoxide production did not always increase calcium in A1/V1+ neurons, suggesting a critical role of an unknown factor. CCCP caused both superoxide production and mitochondrial depolarization but neither correlated with calcium fluxes in A1/V1+ neurons. Rotenone-induced calcium responses in 'responding' A1/V1+ neurons correlated with mitochondrial depolarization but not superoxide production. Our data are consistent with the hypothesis that mitochondrial dysfunction causes calcium fluxes in a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles

PubMed Central

Xu, Bing; Fu, Ying; Liu, Yan; Agvanian, Sosse; Wirka, Robert C.; Baum, Rachel; Zhou, Kang; Shaw, Robin M.

2017-01-01

Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood. PMID:28806752

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

PubMed

Xu, Bing; Fu, Ying; Liu, Yan; Agvanian, Sosse; Wirka, Robert C; Baum, Rachel; Zhou, Kang; Shaw, Robin M; Hong, TingTing

2017-08-01

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes.

PubMed

Taruno, Akiyuki; Vingtdeux, Valérie; Ohmoto, Makoto; Ma, Zhongming; Dvoryanchikov, Gennady; Li, Ang; Adrien, Leslie; Zhao, Haitian; Leung, Sze; Abernethy, Maria; Koppel, Jeremy; Davies, Peter; Civan, Mortimer M; Chaudhari, Nirupa; Matsumoto, Ichiro; Hellekant, Göran; Tordoff, Michael G; Marambaud, Philippe; Foskett, J Kevin

2013-03-14

Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways. However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel, is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception.

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

PubMed

Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

2015-09-22

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Acute administration of tumour necrosis factor-α induces spontaneous calcium release via the reactive oxygen species pathway in atrial myocytes.

PubMed

Zuo, Song; Li, Lin-Ling; Ruan, Yan-Fei; Jiang, Le; Li, Xin; Li, Song-Nan; Wen, Song-Nan; Bai, Rong; Liu, Nian; Du, Xin; Dong, Jian-Zeng; Ma, Chang-Sheng

2017-10-17

The arrhythmogenic mechanisms of atrial fibrillation (AF) that are induced by acute inflammation, such as postoperative AF, are not well understood. We investigated the acute effects of tumour necrosis factor-α (TNF-α) that mimic acute inflammation on Ca2+ handling in isolated atrial myocytes and its underlying mechanisms. Cytosol Ca2+ handling and mitochondrial reactive oxygen species (ROS) production were studied in freshly isolated atrial myocytes of wild-type mice that were exposed to TNF-α (0.05 ng/mL) for 2 h by Ionoptix and confocal microscopy. The acute effects of TNF-α on Ca2+ handling were decreased amplitudes and prolonged decay times of Ca2+ transients in isolated atrial myocytes. A significant reduction in the sarcoplasmic reticulum (SR) Ca2+ content was detected in TNF-α treated cells, which was associated with increased spontaneous Ca2+ release events. In particular, physiological concentrations of TNF-α dramatically promoted the frequency of spontaneous Ca2+ waves and Ca2+ sparks, while the spark mass presented with reduced amplitudes and prolonged durations. The underlying mechanisms of pro-arrhythmic effects of TNF-α were further investigated. Acute exposure to TNF-α rapidly promoted mitochondrial ROS production that was correlated with the acute effect of TNF-α on Ca2+ handling, and enhanced the oxidation of calcium/calmodulin-dependent protein kinase II (CaMKII) and the phosphorylation of RyR2. However, the performance of ROS inhibitor, DL-Dithiothreitol (DTT), reversed Ca2+ handling disorders induced by TNF-α. Tumour necrosis factor-α rapidly increases spontaneous Ca2+ release and promotes atrial arrhythmogenesis via the ROS pathway, which suggests that antioxidant therapy is a promising strategy for acute inflammation related AF. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

Adsorption and release of amino acids mixture onto apatitic calcium phosphates analogous to bone mineral

NASA Astrophysics Data System (ADS)

El Rhilassi, A.; Mourabet, M.; El Boujaady, H.; Bennani-Ziatni, M.; Hamri, R. El; Taitai, A.

2012-10-01

Study focused on the interaction of adsorbate with poorly crystalline apatitic calcium phosphates analogous to bone mineral. Calcium phosphates prepared in water-ethanol medium at physiological temperature (37 °C) and neutral pH, their Ca/P ratio was between 1.33 and 1.67. Adsorbate used in this paper takes the mixture form of two essential amino acids L-lysine and DL-leucine which have respectively a character hydrophilic and hydrophobic. Adsorption and release are investigated experimentally; they are dependent on the phosphate type and on the nature of adsorbate L-lysine, DL-leucine and their mixture. Adsorption of mixture of amino acids on the apatitic calcium phosphates is influenced by the competition between the two amino acids: L-lysine and DL-leucine which exist in the medium reaction. The adsorption kinetics is very fast while the release kinetics is slow. The chemical composition of apatite has an influence on both adsorption and release. The interactions adsorbate-adsorbent are electrostatic type. Adsorption and release reactions of the amino acid mixture are explained by the existence of the hydrated surface layer of calcium phosphate apatite. The charged sbnd COOsbnd and sbnd NH3+ of adsorbates are the strongest groups that interact with the surface of apatites, the adsorption is mainly due to the electrostatic interaction between the groups sbnd COOsbnd of amino acids and calcium Ca2+ ions of the apatite. Comparative study of interactions between adsorbates (L-lysine, DL-leucine and their mixture) and apatitic calcium phosphates is carried out in vitro by using UV-vis and infrared spectroscopy IR techniques.

The Effects of Electrical Stimuli on Calcium Change and Histamine Release in Rat Basophilic Leukemia Mast Cells

NASA Astrophysics Data System (ADS)

Zhu, Dan; Wu, Zu-Hui; Chen, Ji-Yao; Zhou, Lu-Wei

2013-06-01

We apply electric fields at different frequencies of 0.1, 1, 10 and 100 kHz to the rat basophilic leukemia (RBL) mast cells in calcium-containing or calcium-free buffers. The stimuli cause changes of the intracellular calcium ion concentration [Ca2+]i as well as the histamine. The [Ca2+]i increases when the frequency of the external electric field increases from 100 Hz to 10 kHz, and then decreases when the frequency further increases from 10 kHz to 100 kHz, showing a peak at 100 kHz. A similar frequency dependence of the histamine release is also found. The [Ca2+]i and the histamine releases at 100 Hz are about the same as the values of the control group with no electrical stimulation. The ruthenium red (RR), an inhibitor to the TRPV (transient receptor potential (TRP) family V) channels across the cell membrane, is used in the experiment to check whether the electric field stimuli act on the TRPV channels. Under an electric field of 10 kHz, the [Ca2+]i in a calcium-concentration buffer is about 3.5 times as much as that of the control group with no electric stimulation, while the [Ca2+]i in a calcium-free buffer is only about 2.2 times. Similar behavior is also found for the histamine release. RR blockage effect on the [Ca2+]i decrease is statistically significant (~75%) when mast cells in the buffer with calcium are stimulated with a 10 kHz electric field in comparison with the result without the RR treatment. This proves that TRPVs are the channels that calcium ions inflow through from the extracellular environment under electrical stimuli. Under this condition, the histamine is also released following a similar way. We suggest that, as far as an electric stimulation is concerned, an application of ac electric field of 10 kHz is better than other frequencies to open TRPV channels in mast cells, and this would cause a significant calcium influx resulting in a significant histamine release, which could be one of the mechanisms for electric therapy.

Reciprocal Regulation of Reactive Oxygen Species and Phospho-CREB Regulates Voltage Gated Calcium Channel Expression during Mycobacterium tuberculosis Infection

PubMed Central

Selvakumar, Arti; Antony, Cecil; Singhal, Jhalak; Tiwari, Brijendra K.; Singh, Yogendra; Natarajan, Krishnamurthy

2014-01-01

Our previous work has demonstrated the roles played by L-type Voltage Gated Calcium Channels (VGCC) in regulating Mycobacterium tuberculosis (M. tb) survival and pathogenesis. Here we decipher mechanisms and pathways engaged by the pathogen to regulate VGCC expression in macrophages. We show that M. tb and its antigen Rv3416 use phospho-CREB (pCREB), Reactive Oxygen Species (ROS), Protein Kinase C (PKC) and Mitogen Activated Protein Kinase (MAPK) to modulate VGCC expression in macrophages. siRNA mediated knockdown of MyD88, IRAK1, IRAK2 or TRAF6 significantly inhibited antigen mediated VGCC expression. Inhibiting Protein Kinase C (PKC) or MEK-ERK1/2 further increased VGCC expression. Interestingly, inhibiting intracellular calcium release upregulated antigen mediated VGCC expression, while inhibiting extracellular calcium influx had no significant effect. siRNA mediated knockdown of transcription factors c-Jun, SOX5 and CREB significantly inhibited Rv3416 mediated VGCC expression. A dynamic reciprocal cross-regulation between ROS and pCREB was observed that in turn governed VGCC expression with ROS playing a limiting role in the process. Further dissection of the mechanisms such as the interplay between ROS and pCREB would improve our understanding of the regulation of VGCC expression during M. tb infection. PMID:24797940

PRESENILIN-NULL CELLS HAVE ALTERED TWO-PORE CALCIUM CHANNEL EXPRESSION AND LYSOSOMAL CALCIUM; IMPLICATIONS FOR LYSOSOMAL FUNCTION

PubMed Central

Kayala, Kara M Neely; Dickinson, George D; Minassian, Anet; Walls, Ken C; Green, Kim N; LaFerla, Frank M

2012-01-01

Presenilins are necessary for calcium homeostasis and also for efficient proteolysis through the autophagy/lysosome system. Presenilin regulates both endoplasmic reticulum calcium stores and autophagic proteolysis in a γ-secretase independent fashion. The endo-lysosome system can also act as a calcium store, with calcium efflux channels being recently identified as two-pore channels 1 and 2. Here we investigated lysosomal calcium content and the channels that mediate calcium release from these acidic stores in presenilin knockout cells. We report that presenilin loss leads to a lower total lysosomal calcium store despite the buildup of lysosomes found in these cells. Additionally, we find alterations in two-pore calcium channel protein expression, with loss of presenilin preventing the formation of a high molecular weight species of TPC1 and TPC2. Finally, we find that treatments that disturb lysosomal calcium release lead to a reduction in autophagy function yet lysosomal inhibitors do not alter two-pore calcium channel expression. These data indicate that alterations in lysosomal calcium in the absence of presenilins might be leading to disruptions in autophagy. PMID:23103503

Use of calcium caseinate in association with lecithin for masking the bitterness of acetaminophen--comparative study with sodium caseinate.

PubMed

Hoang Thi, Thanh Huong; Lemdani, Mohamed; Flament, Marie-Pierre

2013-11-18

Owing to a variety of structural and functional properties, milk proteins are steadily studied for food and pharmaceutical applications. In the present study, calcium caseinate in association with lecithin was firstly investigated in order to encapsulate the acetaminophen through spray-drying for taste-masking purpose for pediatric medicines. A 2(4)-full factorial design revealed that the spray flow, the calcium caseinate amount and the lecithin amount had significant effects on the release of drug during the first 2 min. Indeed, increasing the spray flow and/or the calcium caseinate amount led to increase the released amount, whereas increasing the lecithin amount decreased the released amount. The "interaction" between the calcium caseinate amount and the lecithin amount was also shown to be statistically significant. The second objective was to compare the efficiency of two caseinate-based formulations, i.e. sodium caseinate and calcium caseinate, on the taste-masking effect. The characteristics of spray-dried powders determined by SEM and DSC were shown to depend on the caseinate/lecithin proportion rather than the type of caseinate. Interestingly, calcium caseinate-based formulations were found to lower the released amount of drug during the early time to a higher extent than sodium caseinate-based formulations, which indicates better taste-masking efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Local Control Models of Cardiac Excitation–Contraction Coupling

PubMed Central

Stern, Michael D.; Song, Long-Sheng; Cheng, Heping; Sham, James S.K.; Yang, Huang Tian; Boheler, Kenneth R.; Ríos, Eduardo

1999-01-01

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation–contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation–contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution. PMID:10051521

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

PubMed

Chang, Kai-Wei; Huang, Nancy A; Liu, I-Hsuan; Wang, Yi-Hui; Wu, Ping; Tseng, Yen-Tzu; Hughes, Michael W; Jiang, Ting Xin; Tsai, Mong-Hsun; Chen, Chien-Yu; Oyang, Yen-Jen; Lin, En-Chung; Chuong, Cheng-Ming; Lin, Shau-Ping

2015-01-23

Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.

Calcium and 1,25-dihydroxyvitamin D3 modulate genes of immune and inflammatory pathways in the human colon: a human crossover trial.

PubMed

Protiva, Petr; Pendyala, Swaroop; Nelson, Celeste; Augenlicht, Leonard H; Lipkin, Martin; Holt, Peter R

2016-05-01

A high dietary calcium intake with adequate vitamin D status has been linked to lower colorectal cancer risk, but the mechanisms of these effects are poorly understood. The objective of this study was to elucidate the effects of a Western-style diet (WD) and supplemental calcium and/or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the colorectal mucosa. We conducted 2 crossover trials to define molecular pathways in the human colorectum altered by 1) a 4-wk WD supplemented with and without 2 g calcium carbonate/d and 2) a 4-wk WD supplemented with 1,25(OH)2D3 (0.5 μg/d) with or without 2 g calcium carbonate/d. The primary study endpoint was genome-wide gene expression in biopsy specimens of the rectosigmoid colonic mucosa. Serum and urinary calcium concentrations were also measured. Changes in urinary calcium accurately reflected calcium consumption. The WD induced modest upregulation of genes involved in inflammatory pathways, including interferon signaling, and calcium supplementation reversed these toward baseline. In contrast, supplementation of the WD with 1,25(OH)2D3 induced striking upregulation of genes involved in inflammation, immune response, extracellular matrix, and cell adhesion. Calcium supplementation largely abrogated these changes. Supplementing 1,25(OH)2D3 to a WD markedly upregulated genes in immune response and inflammation pathways, which were largely reversed by calcium supplementation. This study provides clinical trial evidence of global gene expression changes occurring in the human colorectum in response to calcium and 1,25(OH)2D3 intervention. One action of 1,25(OH)2D3 is to upregulate adaptive immunity. Calcium appears to modulate this effect, pointing to its biological interaction in the mucosa. This trial was registered at clinicaltrials.gov as NCT00298545 Trial protocol is available at http://clinicalstudies.rucares.org (protocol numbers PHO475 and PHO554). © 2016 American Society for Nutrition.

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

PubMed

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.

Ion release from, and fluoride recharge of a composite with a fluoride-containing bioactive glass.

PubMed

Davis, Harry B; Gwinner, Fernanda; Mitchell, John C; Ferracane, Jack L

2014-10-01

Materials that are capable of releasing ions such as calcium and fluoride, that are necessary for remineralization of dentin and enamel, have been the topic of intensive research for many years. The source of calcium has most often been some form of calcium phosphate, and that for fluoride has been one of several metal fluoride or hexafluorophosphate salts. Fluoride-containing bioactive glass (BAG) prepared by the sol-gel method acts as a single source of both calcium and fluoride ions in aqueous solutions. The objective of this investigation was to determine if BAG, when added to a composite formulation, can be used as a single source for calcium and fluoride ion release over an extended time period, and to determine if the BAG-containing composite can be recharged upon exposure to a solution of 5000ppm fluoride. BAG 61 (61% Si; 31% Ca; 4% P; 3% F; 1% B) and BAG 81 (81% Si; 11% Ca; 4% P; 3% F; 1% B) were synthesized by the sol-gel method. The composite used was composed of 50/50 Bis-GMA/TEGDMA, 0.8% EDMAB, 0.4% CQ, and 0.05% BHT, combined with a mixture of BAG (15%) and strontium glass (85%) to a total filler load of 72% by weight. Disks were prepared, allowed to age for 24h, abraded, then placed into DI water. Calcium and fluoride release was measured by atomic absorption spectroscopy and fluoride ion selective electrode methods, respectively, after 2, 22, and 222h. The composite samples were then soaked for 5min in an aqueous 5000ppm fluoride solution, after which calcium and fluoride release was again measured at 2, 22, and 222h time points. Prior to fluoride recharge, release of fluoride ions was similar for the BAG 61 and BAG 81 composites after 2h, and also similar after 22h. At the four subsequent time points, one prior to, and three following fluoride recharge, the BAG 81 composite released significantly more fluoride ions (p<0.05). Both composites were recharged by exposure to 5000ppm fluoride, although the BAG 81 composite was recharged more than the BAG 61 composite. The BAG 61 composite released substantially more calcium ions prior to fluoride recharge during each of the 2 and 22h time periods. Thereafter, the release of calcium at the four subsequent time points was not significantly different (p>0.05) for the two composites. These results show that, when added to a composite formulation, fluoride-containing bioactive glass made by the sol-gel route can function as a single source for both calcium and fluoride ions, and that the composite can be readily recharged with fluoride. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells.

PubMed

Knight, Derek E

2002-04-01

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca2+-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised 125I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of 125I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered 125I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered 125I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 mm for 125I-transferrin and 1.0 mm for catecholamine, and the intracellular concentrations were 0.1 microm and 1 microm, respectively. There was significant 125I-transferrin recycling in the virtual absence of intracellular Ca2+, but the rate increased when Ca2+ was raised above 1 nm, and peaked at 1 microm when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.

Neuropeptide Secreted from a Pacemaker Activates Neurons to Control a Rhythmic Behavior

PubMed Central

Wang, Han; Girskis, Kelly; Janssen, Tom; Chan, Jason P.; Dasgupta, Krishnakali; Knowles, James A.; Schoofs, Liliane; Sieburth, Derek

2013-01-01

Summary Background Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 seconds, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown. Results Here we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We find that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagmin. We identify AEX-2, the G protein-coupled receptor on the GABAergic neurons, as the receptor of NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-40-derived peptide depolarizes the GABAergic neurons in vivo. Conclusions Our results show that NLP-40 carries the timing information from the pacemaker via calcium-dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors. PMID:23583549

Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

PubMed Central

Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2014-01-01

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

Mechanism of ipamorelin-evoked insulin release from the pancreas of normal and diabetic rats.

PubMed

Adeghate, Ernest; Ponery, Abdul Samad

2004-12-01

To examine the effect of ipamorelin (IPA), a novel pentapeptide with a strong growth hormone releasing potency, on insulin secretion from pancreatic tissue fragments of normal and diabetic rats. Diabetes mellitus was induced by streptozotocin (60 mg kg(-1)). Four weeks after the induction of diabetes, pancreatic tissue fragments of normal and diabetic rats were removed and incubated with different concentrations (10(-12) - 10(-6) M) of IPA. Insulin release from the pancreas was measured by radioimmunoassay. Ipamorelin evoked significant (p<0.04) increases in insulin secretion from the pancreas of normal and diabetic rats. Either diltiazem or yohimbine or propranolol or a combination of atropine, propranolol and yohimbine inhibited IPA-evoked insulin secretion significantly (p<0.03) from the pancreas of normal and diabetic rats. Atropine caused a significant (p<0.007) reduction in the IPA-induced insulin secretion in diabetic but not in normal rats. IPA stimulates insulin release through the calcium channel and the adrenergic receptor pathways. This is the first study to examine the effect of ipamorelin on insulin secretion in the pancreas.

Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling

PubMed Central

Dickson, Eamonn J.; Falkenburger, Björn H.

2013-01-01

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 (<1%). These differences can be attributed purely to differences in receptor abundance. Full amplitude 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. PMID:23630337

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

PubMed

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 (<1%). These differences can be attributed purely to differences in receptor abundance. Full amplitude 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.

Kinetic Studies of Calcium-Induced Calcium Release in Cardiac Sarcoplasmic Reticulum Vesicles

PubMed Central

Sánchez, Gina; Hidalgo, Cecilia; Donoso, Paulina

2003-01-01

Fast Ca2+ release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca2+. Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca2+] and [Mg2+]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa ≤5. These results suggest that channels respond differently when exposed to sudden [Ca2+] changes than when exposed to Ca2+ for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg2+ produced a marked inhibition of release kinetics at pCa 6 (K0.5 = 63 μM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg2+] tested and increased at pCa 6 the K0.5 for Mg2+ inhibition, from 63 μM to 136 μM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca2+ and Mg2+ followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing. PMID:12668440

Calcium release rates from tooth enamel treated with dentifrices containing whitening agents and abrasives.

PubMed

Araujo, Danilo Barral; Silva, Luciana Rodrigues; de Araujo, Roberto Paulo Correia

2010-01-01

Tooth whitening agents containing hydrogen peroxide and carbamide peroxide are used frequently in esthetic dental procedures. However, lesions on the enamel surface have been attributed to the action of these products. Using conventional procedures for separating and isolating biological structures, powdered enamel was obtained and treated with hydrogen peroxide, carbamide peroxide, and sodium bicarbonate, ingredients typically found in dentifrices. The enamel was exposed to different pH levels, and atomic emission spectrometry was used to determine calcium release rates. As the pH level increased, the rate of calcium release from enamel treated with dentifrices containing whitening agents decreased. Carbamide peroxide produced the lowest amount of decalcification, while sodium bicarbonate produced the highest release rates at all pH levels.

Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes.

PubMed

Moulian, N; Gaudry-Talarmain, Y M

1993-06-01

The presence of P-type calcium channels in synaptosomes prepared from electric organ of Torpedo marmorata was investigated by using the venom of Agelenopsis aperta, a toxin purified from it, FTX, and its synthetic analog. We analysed the action of these agents on acetylcholine release which was continuously followed using a chemiluminescent assay. Agelenopsis aperta venom, FTX and synthetic FTX inhibit acetylcholine release from synaptosomes induced by a presynaptic membrane depolarization with 60 mM KCl. A stronger inhibition of acetylcholine release was observed with the venom than with FTX (70 and 50%, respectively). Another way of triggering acetylcholine release from Torpedo synaptosomes is to insert in the presynaptic membrane a calcium ionophore A23187 which allows the bypass of the natural calcium channels. The venom of Agelenopsis aperta inhibits A23187-evoked acetylcholine release. Purified and synthetic FTX does not possess this property, suggesting that this inhibition of acetylcholine release was due to other toxins of the venom. Another type of pharmacological sensitivity of Torpedo calcium channels was also demonstrated using omega-conotoxin GVIA. At a concentration of 20 microM, this toxin was able to inhibit about 35% of KCl-evoked acetylcholine release. When FTX + omega-conotoxin GVIA were applied together, the inhibitory effect on KCl-evoked acetylcholine release was not significantly increased in comparison with the one observed with FTX alone. In conclusion, we examined the effect of different agents on acetylcholine release from Torpedo marmorata electric organ synaptosomes; acetylcholine release was elicited with KCl depolarization and followed continuously with a chemiluminescent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper amplification of prostaglandin E/sub 2/ stimulation of the release of luteinizing hormone-releasing hormone is a postreceptor event

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnea, A.; Cho, G.

1987-01-01

The authors have shown that copper amplifies prostaglandin E/sub 2/ (PGE/sub 2/) stimulation of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA) and that this process is calcium-dependent. Since a Ca-cAMP pathway has been implicated in PGE/sub 2/ action on the LH-RH neuron, in this study the authors wished to ascertain if copper exerts its effect on the PGE/sub 2/ receptor or on a postreceptor component involved in PGE/sub 2/ action. MEA of adult male rats were incubated for 5 min with 200 ..mu..M Cu/histidine and then incubated for 15 min either with 10 ..mu..M PGE/submore » 2/ (Cu/PGE/sub 2/), 100 ..mu..M forskolin (Cu/forskolin), or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (Cu/cAMP). Basal release of LH-RH was 4.6 +/- 0.45 pg/15 min per MEA determined by radioimmunoassay. Net stimulated release during the 15-min exposure to PGE/sub 2/, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate was 3.6 +/- 0.52, 3.1 +/- 0.39, and 1.6 +/- 0.42 pg/15 min per MEA, respectively. Net stimulated release after exposure to Cu/PGE/sub 2/, Cu/forskolin, or Cu/cAMP indicated that copper amplifies the action of PGE/sub 2/ and forskolin but not cAMP action. When MEA were exposed to a mixture of PGE/sub 2/ and forskolin for 15 min, the effects of these two secretagogues on LH-RH release were not additive. In contrast to PGE/sub 2/ and forskolin, copper did not amplify K/sup +/ stimulation of OH-RH release. These results are supportive of the proposition that PGE/sub 2/ stimulation of OH-RH release is mediated by the Ca-cAMP pathway and that copper amplification of PGE/sub 2/ action is a postreceptor event.« less

Promotion of neuritogenesis in mouse neuroblastoma cells by ganglioside GM3: Involvement of three signal pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, A.H.

1989-01-01

Ganglioside GM3 was extracted from human placentae and tested for neuritogenic properties towards the mouse neuroblastoma cell line Neuro-2A. GM3 (2.5 {mu}M) was found to inhibit cell growth when added exogenously to the cell culture. ({sup 3}H)Thymidine incorporation was inhibited by 49% within 6 hr. Neuritogenesis was evident within 24 hr evidenced by an increase in the number and length of neurites produced compared to control cells. An enzymatic assay for protein kinase C activity was employed to study effects of GM3 on the subcellular localization of the enzyme. Ganglioside GM3 was found to alter the subcellular localization of themore » phospholipid- and calcium-dependent protein kinase C. These results were confirmed using a binding assay employing the labeled phorbol ester ({sup 3}H)phorbol-12,13-dibutyrate. Finally, GM3-modulation of IP{sub 3} formation and cytosolic calcium in the Neuro-2A cells was investigated. GM3 did not alter the phosphoinositol metabolism as evidenced by IP{sub 3} formation in these cells. However, the addition of GM3 (16 {mu}M) to cells loaded with the photoprotein, aequorin, induced an increase in the intracellular calcium concentration within 2 min, which was sustained for 10 min. Removal of external calcium by chelation did not abrogate the response to GM3, indicating that calcium was being released from internal stores. The calcium influx was temporally correlated with the translocation of protein kinase C, providing a rationale whereby GM3 may induce the enzyme to translocate.« less

FGF2 activates TRPC and Ca2+ signaling leading to satellite cell activation

PubMed Central

Liu, Yewei; Schneider, Martin F.

2013-01-01

Satellite cells, as stem cells of adult skeletal muscle, are tightly associated with the differentiated muscle fibers and remain quiescent in the absence of muscle damage. In response to an injury, the quiescent satellite cell is activated by soluble factors, including FGFs released from injured myofibers. Using immunostaining, we here first show that TRPC1 channels are highly expressed in satellite cells attached to muscle fibers. Since CD34, a traditional stem cell marker, was recently found to be expressed in skeletal muscle satellite cells we labeled living satellite cells in their physiological niche associated with host FDB fibers using anti-CD34-FITC antibody. We then monitored intra-cellular calcium in anti-CD34-FITC labeled satellite cells attached to muscle fibers using the calcium sensitive dye X rhod-1 which has little fluorescence cross talk with FITC. FGF2 increased intracellular calcium in satellite cells, which was antagonized by the TRPC channel blocker SKF 96365. Immunostaining showed that NFATc3 is highly expressed in satellite cells, but not in host FDB fibers. Elevation of intracellular calcium by FGF2 is accompanied by nuclear translocation of NFATc3 and NFATc2 and by an increase in the number of MyoD positive cells per muscle fiber, both of which were attenuated by TRPC blocker SKF 96365. Our results suggest a novel pathway of satellite cell activation where FGF2 enhances calcium influx through a TRPC channel, and the increased cytosolic calcium leads to both NFATc3 and NFATc2 nuclear translocation and enhanced number of MyoD positive satellite cells per muscle fiber. PMID:24575047

Physicochemical properties of calcium silicate-based formulations MTA Repair HP and MTA Vitalcem.

PubMed

Guimarães, Bruno Martini; Prati, Carlo; Duarte, Marco Antonio Hungaro; Bramante, Clovis Monteiro; Gandolfi, Maria Giovanna

2018-04-05

This study aimed to analyze the following physicochemical properties: radiopacity, final setting time, calcium release, pH change, solubility, water sorption, porosity, surface morphology, and apatite-forming ability of two calcium silicate-based materials. We tested MTA Repair HP and MTA Vitalcem in comparison with conventional MTA, analyzing radiopacity and final setting time. Water absorption, interconnected pores and apparent porosity were measured after 24-h immersion in deionized water at 37°C. Calcium and pH were tested up to 28 d in deionized water. We analyzed data using two-way ANOVA with Student-Newman-Keuls tests (p<0.05). We performed morphological and chemical analyses of the material surfaces using ESEM/EDX after 28 d in HBSS. MTA Repair HP showed similar radiopacity to that of conventional MTA. All materials showed a marked alkalinizing activity within 3 h, which continued for 28 d. MTA Repair HP showed the highest calcium release at 28 d (p<0.05). MTA Vitalcem showed statistically higher water sorption and solubility values (p<0.05). All materials showed the ability to nucleate calcium phosphate on their surface after 28 d in HBSS. MTA Repair HP and MTA Vitalcem had extended alkalinizing activity and calcium release that favored calcium phosphate nucleation. The presence of the plasticizer in MTA HP might increase its solubility and porosity. The radiopacifier calcium tungstate can be used to replace bismuth oxide.

Beyond the Channel: Metabotropic Signaling by Nicotinic Receptors.

PubMed

Kabbani, Nadine; Nichols, Robert A

2018-04-01

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel (LGIC) that plays an important role in cellular calcium signaling and contributes to several neurological diseases. Agonist binding to the α7 nAChR induces fast channel activation followed by inactivation and prolonged desensitization while triggering long-lasting calcium signaling. These activities foster neurotransmitter release, synaptic plasticity, and somatodendritic regulation in the brain. We discuss here the ability of α7 nAChRs to operate in ionotropic (α7 i ) and metabotropic (α7 m ) modes, leading to calcium-induced calcium release (CICR) and G protein-associated inositol trisphosphate (IP 3 )-induced calcium release (IICR), respectively. Metabotropic activity extends the spatial and temporal aspects of calcium signaling by the α7 channel beyond its ionotropic limits, persisting into the desensitized state. Delineation of the ionotropic and metabotropic properties of the α7 nAChR will provide definitive indicators of moment-to-moment receptor functional status that will, in turn, spearhead new drug development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rechargeable calcium phosphate orthodontic cement with sustained ion release and re-release

NASA Astrophysics Data System (ADS)

Zhang, Ling; Weir, Michael D.; Chow, Laurence C.; Reynolds, Mark A.; Xu, Hockin H. K.

2016-11-01

White spot lesions (WSL) due to enamel demineralization are major complications for orthodontic treatments. Calcium phosphate (CaP) dental resins with Ca and P ion releases are promising for remineralization. However, previous Ca and P releases lasted for only weeks. Experimental orthodontic cements were developed using pyromellitic glycerol dimethacrylate (PMGDM) and ethoxylated bisphenol A dimethacrylate (EBPADMA) at mass ratio of 1:1 (PE); and PE plus 10% of 2-hydroxyethyl methacrylate (HEMA) and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (PEHB). Particles of amorphous calcium phosphate (ACP) were incorporated into PE and PEHB at 40% filler level. Specimens were tested for bracket-enamel shear bond strength, water sorption, CaP release, and ion recharge and re-release. PEHB+40ACP had higher bracket-enamel bond strength and ion release and rechargeability than PE+40ACP. ACP incorporation into the novel orthodontic cement did not adversely affect the bracket-enamel bond strength. Ion release and re-release from the novel ACP orthodontic cement indicated favorable release and re-release patterns. The recharged orthodontic cement could release CaP ions continuously for four weeks without further recharge. Novel rechargeable orthodontic cement containing ACP was developed with a high bracket-enamel bond strength and the ability to be repeatedly recharged to maintain long-term high levels of CaP ion releases.

Parallel temperature dependence of contracture-associated enzyme release due to anoxia, 2,4-dinitrophenol (DNP), or caffeine and the calcium paradox.

PubMed Central

Ganote, C. E.; Sims, M. A.

1984-01-01

Hypothermia during calcium-free perfusion of hearts protects them from injury caused by subsequent calcium repletion at 37 C (calcium paradox). Injury to calcium-free hearts is also associated with contracture caused by anoxia, 2,4-dinitrophenol (DNP), or caffeine. This study was done for the purpose of determining whether hypothermia during calcium-free perfusions protects hearts from contracture-associated injury. Langendorff-perfused rat hearts were studied in four experimental groups: I) Anoxia: Thirty minutes of anoxic perfusion at 37 C was followed by thirty minutes of anoxic calcium-free perfusion at 37-18 C. II) Calcium paradox: Five minutes of calcium-free perfusion at 37-18 C was followed by calcium repletion at 37 C. III, IVa) Caffeine or DNP: Five minutes of calcium-free perfusion at 37-18 C was followed by addition of 10 mM caffeine or 1 mM DNP in calcium-free medium at 37 C or, IVb) 1 mM DNP in calcium-free medium at 22 C. Injury was assessed by measurement of serial releases of creatine kinase (CK) in effluents and by cellular morphology. The results show that progressive hypothermia to 22 C during calcium-free perfusion periods produced a progressive reduction of CK release and morphologic evidence of injury due to anoxia, caffeine, or DNP, which closely paralleled protection of hearts from the calcium paradox. Protection from injury in all experimental groups was associated with preservation of sarcolemmal membrane integrity and prevention of cell separations at intercalated disk junctions. It is proposed that weakening of intercalated disks occurs during calcium-free perfusions and may be a cause of mechanical fragility of the sarcolemma. Hypothermia may protect hearts from contracture-associated injury by preserving the integrity of intercalated disk junctions during periods of extracellular calcium depletion. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:6742111

Evaluation of Gum of Moringa oleifera as a Binder and Release Retardant in Tablet Formulation

PubMed Central

Panda, D. S.; Choudhury, N. S. K.; Yedukondalu, M.; Si, S.; Gupta, R.

2008-01-01

The present study was undertaken to find out the potential of gum from Moringa oleifera to act as a binder and release retardant in tablet formulations. The effect of calcium sulphate dihydrate (water insoluble) and lactose (water soluble) diluent on the release of propranolol hydrochloride was studied. The DSC thermograms of drug, gum and mixture of gum/drug indicated no chemical interaction. Tablets (F1, F2, F3, and F4) were prepared containing calcium sulphate dihydrate as diluent, propranolol hydrochloride as model drug using 10%, 8%, 6% and 4% w/v of gum solution as binder. Magnesium stearate was used as lubricant. Physical and technological properties of granules and tablets like flow rate, Carr index, Hausner ratio, angle of repose, hardness, friability and disintegration time were determined and found to be satisfactory. Tablets were prepared by wet granulation method containing calcium sulphate dihydrate as excipient, propranolol hydrochloride as model drug using 10%, 20% and 30% of gum as release retardant, magnesium stearate was used as lubricant. Similarly tablets were prepared replacing lactose with calcium sulphate dihydrate. Despite of the widely varying physico-chemical characteristics of the excipients, the drug release profiles were found to be similar. The drug release increased with increasing proportions of the excipient and decreased proportion of the gum irrespective of the solubility characteristics of the excipient. The values of release exponent ‘n’ are between 0.37 and 0.54. This implies that the release mechanism is Fickian. There is no evidence that the dissolution or erosion of the excipient has got any effect on the release of the drug. The t50% values for tablets containing calcium sulphate dihydrate were on an average 10%-15% longer than the tablets containing lactose as excipient. These relatively small differences in t50% values suggest that the nature of excipient used appeared to play a minor role in regulating the release, while the gum content was a major factor. PMID:21394258

Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies.

PubMed

Kofron, Michelle D; Laurencin, Cato T

2004-06-01

One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide-co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway.

PubMed

Haustein, Martin D; Kracun, Sebastian; Lu, Xiao-Hong; Shih, Tiffany; Jackson-Weaver, Olan; Tong, Xiaoping; Xu, Ji; Yang, X William; O'Dell, Thomas J; Marvin, Jonathan S; Ellisman, Mark H; Bushong, Eric A; Looger, Loren L; Khakh, Baljit S

2014-04-16

The spatiotemporal activities of astrocyte Ca²⁺ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²⁺ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²⁺ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²⁺ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²⁺ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Proteomic Analysis of Rat Hippocampus under Simulated Microgravity

NASA Astrophysics Data System (ADS)

Wang, Yun; Li, Yujuan; Zhang, Yongqian; Liu, Yahui; Deng, Yulin

It has been found that microgravity may lead to impairments in cognitive functions performed by CNS. However, the exact mechanism of effects of microgravity on the learning and memory function in animal nervous system is not elucidated yet. Brain function is mainly mediated by membrane proteins and their dysfunction causes degeneration of the learning and memory. To induce simulated microgravity, the rat tail suspension model was established. Comparative O (18) labeling quantitative proteomic strategy was applied to detect the differentially expressed proteins in rat brain hippocampus. The proteins in membrane fraction from rat hippocampus were digested by trypsin and then the peptides were separated by off-gel for the first dimension with 24 wells device encompassing the pH range of 3 - 10. An off-gel fraction was subjected into LC-ESI-QTOF in triplicate. Preliminary results showed that nearly 77% of the peptides identified were specific to one fraction. 676 proteins were identified among which 108 proteins were found differentially expressed under simulated microgravity. Using the KOBAS server, many enriched pathways, such as metabolic pathway, synaptic vesicle cycle, endocytosis, calcium signaling pathway, and SNAREs pathway were identified. Furthermore, it has been found that neurotransmitter released by Ca (2+) -triggered synaptic vesicles fusion may play key role in neural function. Rab 3A might inhibit the membrane fusion and neurotransmitter release. The protein alteration of the synaptic vesicle cycle may further explain the effects of microgravity on learning and memory function in rats. Key words: Microgravity; proteomics; synaptic vesicle; O (18) ({}) -labeling

Calcium Domains around Single and Clustered IP3 Receptors and Their Modulation by Buffers

PubMed Central

Rüdiger, S.; Nagaiah, Ch.; Warnecke, G.; Shuai, J.W.

2010-01-01

Abstract We study Ca2+ release through single and clustered IP3 receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca2+ buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP3Rs produces a distinct [Ca2+] scale (0.5–10 μM), which is smaller than channel pore concentrations (>100 μM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca2+ evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals. PMID:20655827

Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells.

PubMed

Shin, Dong-Hyun; Leem, Dong-Gyu; Shin, Ji-Sun; Kim, Joo-Il; Kim, Kyung-Tack; Choi, Sang Yoon; Lee, Myung-Hee; Choi, Jung-Hye; Lee, Kyung-Tae

2018-04-01

Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Compound K-induced ER stress was confirmed through increased phosphorylation of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular Ca 2+ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Cell survival and intracellular Ca 2+ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.

Crosslink between calcium and sodium signalling.

PubMed

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.

Essential Roles of Raf/Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway, YY1, and Ca2+ Influx in Growth Arrest of Human Vascular Smooth Muscle Cells by Bilirubin*

PubMed Central

Stoeckius, Marlon; Erat, Anna; Fujikawa, Tatsuya; Hiromura, Makoto; Koulova, Anna; Otterbein, Leo; Bianchi, Cesario; Tobiasch, Edda; Dagon, Yossi; Sellke, Frank W.; Usheva, Anny

2012-01-01

The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition. PMID:22262839

The Effect of Calcium Phosphate Particle Shape and Size on their Antibacterial and Osteogenic Activity in the Delivery of Antibiotics in vitro

PubMed Central

Uskoković, Vuk; Batarni, Samir Shariff; Schweicher, Julien; King, Andrew; Desai, Tejal A.

2013-01-01

Powders composed of four morphologically different calcium phosphate particles were prepared by precipitation from aqueous solutions: flaky, brick-like, elongated orthogonal, and spherical. The particles were then loaded with either clindamycin phosphate as the antibiotic of choice, or fluorescein, a model molecule used to assess the drug release properties. A comparison was carried out of the comparative effect of such antibiotic-releasing materials on: sustained drug release profiles; Staphylococcus aureus growth inhibition; and osteogenic propensities in vitro. Raman spectroscopic analysis indicated the presence of various calcium phosphate phases, including monetite (flaky and elongated orthogonal particles), octacalcium phosphate (brick-shaped particles) and hydroxyapatite (spherical particles). Testing the antibiotic-loaded calcium phosphate powders for bacterial growth inhibition demonstrated satisfying antibacterial properties both in broths and on agar plates. All four calcium-phosphate-fluorescein powders exhibited sustained drug release over 21 days. The calcium phosphate sample with the highest specific surface area and the smallest, spherical particle size was the most effective in both drug loading and release, consequently having the highest antibacterial efficiency. Moreover, the highest cell viability, the largest gene expression upregulation of three different osteogenic markers – osteocalcin, osteopontin and Runx2 - as well as the least disrupted cell cytoskeleton and cell morphologies were also noticed for the calcium phosphate powder composed of smallest, spherical nanosized particles. Still, all four powders exerted a viable effect on osteoblastic MC3T3-E1 cells in vitro, as evidenced by both morphological assessments on fluorescently stained cells and measurements of their mitochondrial activity. The obtained results suggest that the nanoscale particle size and the corresponding coarseness of the surface of particle conglomerates as the cell attachment points may present a favorable starting point for the development of calcium-phosphate-based osteogenic drug delivery devices. PMID:23484624

Growth hormone-releasing hormone as an agonist of the ghrelin receptor GHS-R1a

PubMed Central

Casanueva, Felipe F.; Camiña, Jesus P.; Carreira, Marcos C.; Pazos, Yolanda; Varga, Jozsef L.; Schally, Andrew V.

2008-01-01

Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1–29)NH2 (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of 125I-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1–42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control. PMID:19088192

Growth hormone-releasing hormone as an agonist of the ghrelin receptor GHS-R1a.

PubMed

Casanueva, Felipe F; Camiña, Jesus P; Carreira, Marcos C; Pazos, Yolanda; Varga, Jozsef L; Schally, Andrew V

2008-12-23

Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1-29)NH(2) (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of (125)I-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1-42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control.

Magnesium sulfate provides neuroprotection in lipopolysaccharide-activated primary microglia by inhibiting NF-κB pathway.

PubMed

Gao, Feng; Ding, Baozhong; Zhou, Longan; Gao, Xueshan; Guo, Huiguang; Xu, Hong

2013-10-01

Magnesium sulfate has been used as an anticonvulsant in severe preeclamptic or eclamptic women prior to surgical trauma, but its effects on neuroinflammation is not well defined. In the present study, we investigated the neuroprotective effects of magnesium sulfate in lipopolysaccharide (LPS)-induced microglia and explored the underlying mechanism. Microglia was incubated with LPS in the presence or absence of various concentrations of magnesium sulfate, or L-type calcium channel activator BAY-K8644. The levels of inflammatory mediators, such as nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α, were measured using enzyme-linked immunosorbent assay. The expression of inducible nitric oxide synthase mRNA was detected by reverse-transcription polymerase chain reaction. Nuclear factor κB (NF-κB) activity in the nuclear extract of microglia was detected by NF-κB p50/p65 transcription factor assay kit. Magnesium sulfate at 5 and 10 mmol/L significantly inhibited the release of nitric oxide, prostaglandin E2, interleukin 1β, and tumor necrosis factor α, and the expression of inducible nitric oxide synthase mRNA in LPS-activated microglia. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in a dose-dependent manner. Notably, these effects were significantly reversed by L-type calcium channel activator BAY-K8644. Magnesium sulfate protects microglia against LPS-induced release of inflammatory mediators, and these effects may be mediated by inhibiting L-type calcium channels and NF-κB signaling. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

PubMed Central

Held, Katharina; Kichko, Tatjana; De Clercq, Katrien; Klaassen, Hugo; Van Bree, Rieta; Vanherck, Jean-Christophe; Marchand, Arnaud; Reeh, Peter W.; Chaltin, Patrick; Voets, Thomas; Vriens, Joris

2015-01-01

Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation. PMID:25733887

Nitric oxide regulation of calcitonin gene-related peptide gene expression in rat trigeminal ganglia neurons

PubMed Central

Bellamy, Jamie; Bowen, Elizabeth J.; Russo, Andrew F.; Durham, Paul L.

2006-01-01

Calcitonin gene-related peptide (CGRP) and nitric oxide are involved in the underlying pathophysiology of migraine and other diseases involving neurogenic inflammation. We have tested the hypothesis that nitric oxide might trigger signaling mechanisms within the trigeminal ganglia neurons that would coordinately stimulate CGRP synthesis and release. Treatment of primary trigeminal ganglia cultures with nitric oxide donors caused a greater than four-fold increase in CGRP release compared with unstimulated cultures. Similarly, CGRP promoter activity was also stimulated by nitric oxide donors and overexpression of inducible nitric oxide synthase (iNOS). Cotreatment with the antimigraine drug sumatriptan greatly repressed nitric oxide stimulation of CGRP promoter activity and secretion. Somewhat surprisingly, the mechanisms of nitric oxide stimulation of CGRP secretion did not require cGMP or PI3-kinase signaling pathways, but rather, nitric oxide action required extracellular calcium and likely involves T-type calcium channels. Furthermore, nitric oxide was shown to increase expression of the active forms of the mitogen-activated protein kinases Jun amino-terminal kinase and p38 but not extracellular signal-related kinase in trigeminal neurons. In summary, our results provide new insight into the cellular mechanisms by which nitric oxide induces CGRP synthesis and secretion from trigeminal neurons. PMID:16630053

Design and characterization of calcium alginate microparticles coated with polycations as protein delivery system.

PubMed

Zarate, J; Virdis, L; Orive, G; Igartua, M; Hernández, R M; Pedraz, J L

2011-01-01

Bovine serum albumin (BSA) loaded calcium alginate microparticles (MPs) produced in this study by a w/o emulsification and external gelation method exhibited spherical and fairly smooth and porous morphology with 1.052 ± 0.057 µm modal particle size. The high permeability of the calcium alginate hydrogel lead to a potent burst effect and too fast protein release. To overcome these problems, MPs were coated with polycations, such as chitosan, poly-L-lysine and DEAE-dextran. Our results demonstrated that coated MPs showed slower release and were able to significantly reduce the release of BSA in the first hour. Therefore, this method can be applied to prepare coated alginate MPs which could be an optimal system for the controlled release of biotherapeutic molecules. Nevertheless, further studies are needed to optimize delivery properties which could provide a sustained release of proteins.

Cavβ2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes.

PubMed

Moreno, Cristian; Hermosilla, Tamara; Morales, Danna; Encina, Matías; Torres-Díaz, Leandro; Díaz, Pablo; Sarmiento, Daniela; Simon, Felipe; Varela, Diego

2015-12-01

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

Dimebon Inhibits Calcium-Induced Swelling of Rat Brain Mitochondria But Does Not Alter Calcium Retention or Cytochrome C Release

PubMed Central

Naga, Kranthi Kumari

2012-01-01

Dimebon was originally introduced as an antihistamine and subsequently investigated as a possible therapeutic for a variety of disorders, including Alzheimer's disease. One putative mechanism underlying the neuroprotective properties of Dimebon is inhibition of mitochondrial permeability transition, based on the observation that Dimebon inhibited the swelling of rat liver mitochondria induced by calcium and other agents that induce permeability transition. Because liver and brain mitochondria differ substantially in their properties and response to conditions associated with opening of the permeability transition pore, we sought to determine whether Dimebon inhibited permeability transition in brain mitochondria. Dimebon reduced calcium-induced mitochondrial swelling but did not enhance the calcium retention capacity or impair calcium-induced cytochrome C release from non-synaptic mitochondria isolated from rat brain cerebral cortex. These findings indicate that Dimebon does not inhibit mitochondrial permeability transition, induced by excessive calcium uptake, in brain mitochondria. PMID:20625939

Dimebon inhibits calcium-induced swelling of rat brain mitochondria but does not alter calcium retention or cytochrome C release.

PubMed

Naga, Kranthi Kumari; Geddes, James W

2011-03-01

Dimebon was originally introduced as an antihistamine and subsequently investigated as a possible therapeutic for a variety of disorders, including Alzheimer's disease. One putative mechanism underlying the neuroprotective properties of Dimebon is inhibition of mitochondrial permeability transition, based on the observation that Dimebon inhibited the swelling of rat liver mitochondria induced by calcium and other agents that induce permeability transition. Because liver and brain mitochondria differ substantially in their properties and response to conditions associated with opening of the permeability transition pore, we sought to determine whether Dimebon inhibited permeability transition in brain mitochondria. Dimebon reduced calcium-induced mitochondrial swelling but did not enhance the calcium retention capacity or impair calcium-induced cytochrome C release from non-synaptic mitochondria isolated from rat brain cerebral cortex. These findings indicate that Dimebon does not inhibit mitochondrial permeability transition, induced by excessive calcium uptake, in brain mitochondria.

Neuropeptide secreted from a pacemaker activates neurons to control a rhythmic behavior.

PubMed

Wang, Han; Girskis, Kelly; Janssen, Tom; Chan, Jason P; Dasgupta, Krishnakali; Knowles, James A; Schoofs, Liliane; Sieburth, Derek

2013-05-06

Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 s, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown. Here, we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We find that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagmin. We identify AEX-2, the G-protein-coupled receptor on the GABAergic neurons, as the receptor for NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-40-derived peptide depolarizes the GABAergic neurons in vivo. Our results show that NLP-40 carries the timing information from the pacemaker via calcium-dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

Calcium phosphate-phosphorylated adenosine hybrid microspheres for anti-osteosarcoma drug delivery and osteogenic differentiation.

PubMed

Zhou, Zi-Fei; Sun, Tuan-Wei; Chen, Feng; Zuo, Dong-Qing; Wang, Hong-Sheng; Hua, Ying-Qi; Cai, Zheng-Dong; Tan, Jun

2017-03-01

Biocompatibility, biodegradability and bioactivity are significantly important in practical applications of various biomaterials for bone tissue engineering. Herein, we develop a functional inorganic-organic hybrid system of calcium phosphate-phosphorylated adenosine (CPPA). Both calcium phosphate and phosphorylated adenosine molecules in CPPA are fundamental components in mammalians and play important roles in biological metabolism. In this work, we report our three leading research qualities: (1) CPPA hybrid microspheres with hollow and porous structure are synthesized by a facile one-step microwave-assisted solvothermal method; (2) CPPA hybrid microspheres show high doxorubicin loading capacity and pH-responsive drug release properties, and demonstrate positive therapeutic effects on six osteosarcoma cell lines in vitro and a mouse model of 143B osteosarcoma subcutaneous tumor in vivo; (3) CPPA hybrid microspheres are favorable to promote osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) by activating the AMPK pathway, with satisfactory evidences from cellular alkaline phosphatase staining, alizarin red staining, real time PCR and western analysis. The as-prepared CPPA hybrid microspheres are promising in anti-osteosarcoma and bone regeneration, which simultaneously display excellent properties on drug delivery and osteogenic differentiation of hBMSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inwardly rectifying potassium channels influence Drosophila wing morphogenesis by regulating Dpp release.

PubMed

Dahal, Giri Raj; Pradhan, Sarala Joshi; Bates, Emily Anne

2017-08-01

Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. Here, we show that inwardly rectifying potassium (Irk) channels regulate release of the Drosophila bone morphogenetic protein Dpp in the developing fly wing and that this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to a broader distribution of Dpp-GFP. Work by others in different cell types has shown that Irk channels regulate peptide release by modulating membrane potential and calcium levels. We found calcium transients in the developing wing, and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, our data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing. © 2017. Published by The Company of Biologists Ltd.

Multitrophic effects of calcium availability on invasive alien plants, birds, and bird prey items

Treesearch

Vince D' Amico; Greg Shriver; Jake Bowman; Meg Ballard; Whitney Wiest; Liz Tymkiw; Melissa Miller

2011-01-01

Acid rain alters forest soil calcium concentrations in two ways: (1) hydrogen ions displace exchangeable calcium adsorbed to soil surfaces, and (2) aluminum is released to soil water by acid rain and displaces adsorbed calcium. This increases the absorption of aluminum by plant roots, and decreases the absorption of calcium, causing calcium to be more readily leached...

Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum

PubMed Central

Echevarría, Wihelma; Leite, M. Fatima; Guerra, Mateus T.; Zipfel, Warren R.; Nathanson, Michael H.

2013-01-01

Calcium is a second messenger in virtually all cells and tissues1. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus. PMID:12717445

Vincristine-sulphate-loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system.

PubMed

Thakkar, Hetal Paresh; Baser, Amit Kumar; Parmar, Mayur Prakashbhai; Patel, Ketul Harshadbhai; Ramachandra Murthy, Rayasa

2012-06-01

Vincristine-sulfate-loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate-coated liposomes show higher cell uptake than uncoated liposomes.

Calcium sources used by post-natal human myoblasts during initial differentiation.

PubMed

Arnaudeau, Serge; Holzer, Nicolas; König, Stéphane; Bader, Charles R; Bernheim, Laurent

2006-08-01

Increases in cytoplasmic Ca(2+) are crucial for inducing the initial steps of myoblast differentiation that ultimately lead to fusion; yet the mechanisms that produce this elevated Ca(2+) have not been fully resolved. For example, it is still unclear whether the increase comes exclusively from membrane Ca(2+) influx or also from Ca(2+) release from internal stores. To address this, we investigated early differentiation of myoblast clones each derived from single post-natal human satellite cells. Initial differentiation was assayed by immunostaining myonuclei for the transcription factor MEF2. When Ca(2+) influx was eliminated by using low external Ca(2+) media, we found that approximately half the clones could still differentiate. Of the clones that required influx of external Ca(2+), most clones used T-type Ca(2+) channels, but others used store-operated channels as influx-generating mechanisms. On the other hand, clones that differentiated in low external Ca(2+) relied on Ca(2+) release from internal stores through IP(3) receptors. Interestingly, by following clones over time, we observed that some switched their preferred Ca(2+) source: clones that initially used calcium release from internal stores to differentiate later required Ca(2+) influx and inversely. In conclusion, we show that human myoblasts can use three alternative mechanisms to increase cytoplasmic Ca(2+) at the onset of the differentiation process: influx through T-types Ca(2+) channels, influx through store operated channels and release from internal stores through IP(3) receptors. In addition, we suggest that, probably because Ca(2+) elevation is essential during initial differentiation, myoblasts may be able to select between these alternate Ca(2+) pathways.

The voltage sensor of excitation–contraction coupling in mammals: Inactivation and interaction with Ca2+

PubMed Central

2017-01-01

In skeletal muscle, the four-helix voltage-sensing modules (VSMs) of CaV1.1 calcium channels simultaneously gate two Ca2+ pathways: the CaV1.1 pore itself and the RyR1 calcium release channel in the sarcoplasmic reticulum. Here, to gain insight into the mechanism by which VSMs gate RyR1, we quantify intramembrane charge movement associated with VSM activation (sensing current) and gated Ca2+ release flux in single muscle cells of mice and rats. As found for most four-helix VSMs, upon sustained depolarization, rodent VSMs lose the ability to activate Ca2+ release channels opening; their properties change from a functionally capable mode, in which the mobile sensor charge is called charge 1, to an inactivated mode, charge 2, with a voltage dependence shifted toward more negative voltages. We find that charge 2 is promoted and Ca2+ release inactivated when resting, well-polarized muscle cells are exposed to low extracellular [Ca2+] and that the opposite occurs in high [Ca2+]. It follows that murine VSMs are partly inactivated at rest, which establishes the reduced availability of voltage sensing as a pathogenic mechanism in disorders of calcemia. We additionally find that the degree of resting inactivation is significantly different in two mouse strains, which underscores the variability of voltage sensor properties and their vulnerability to environmental conditions. Our studies reveal that the resting and activated states of VSMs are equally favored by extracellular Ca2+. Promotion by an extracellular species of two states of the VSM that differ in the conformation of the activation gate requires the existence of a second gate, inactivation, topologically extracellular and therefore accessible from outside regardless of the activation state. PMID:29021148

Synaptic calcium regulation in hair cells of the chicken basilar papilla.

PubMed

Im, Gi Jung; Moskowitz, Howard S; Lehar, Mohammed; Hiel, Hakim; Fuchs, Paul Albert

2014-12-10

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling. Copyright © 2014 the authors 0270-6474/14/3416688-10$15.00/0.

Synaptic Calcium Regulation in Hair Cells of the Chicken Basilar Papilla

PubMed Central

Im, Gi Jung; Moskowitz, Howard S.; Lehar, Mohammed; Hiel, Hakim

2014-01-01

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents (“minis”) resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling. PMID:25505321

Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faddy, Helen M.; Smart, Chanel E.; Xu, Ren

2008-04-09

The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold duringmore » lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.« less

Release of ATP from marginal cells in the cochlea of neonatal rats can be induced by changes in extracellular and intracellular ion concentrations.

PubMed

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

2012-01-01

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. 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. 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 Ca(2+). Furthermore, changes in the concentration of intracellular Ca(2+) induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. 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 A(2).

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

PubMed Central

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

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells

PubMed Central

2010-01-01

Background Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens. Methods We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH3/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERβ, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively). Results All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses. Conclusions Responses mediated by endogenous estrogens representing different life stages are vulnerable to very low concentrations of these structurally related xenoestrogens. Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes. PMID:20950447

Regulation of serotonin release from enterochromaffin cells of rat cecum mucosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simon, C.; Ternaux, J.P.

1990-05-01

The release of endogenous serotonin or previously taken up tritiated serotonin from isolated strips of rat cecum mucosa containing enterochromaffin cells was studied in vitro. Release of tritiated serotonin was increased by potassium depolarization and was decreased by tetrodotoxin, veratridine and the absence of calcium. Endogenous serotonin was released at a lower rate than tritiated serotonin; endogenous serotonin release was stimulated by potassium depolarization but was unaffected by tetrodotoxin, veratridine or the absence of calcium. Carbachol, norepinephrine, clonidine and isoproterenol decreased release of tritiated serotonin but had less or reverse effect on release of endogenous serotonin. The results suggest twomore » different serotoninergic pools within the enterochromaffin cell population.« less

The common inhaled anesthetic isoflurane increases aggregation of huntingtin and alters calcium homeostasis in a cell model of Huntington's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Qiujun; Department of Anesthesiology, The Third Clinical Hospital, Hebei Medical University, Shijiazhuang, Hebei 050051; Liang Ge

2011-02-01

Isoflurane is known to increase {beta}-amyloid aggregation and neuronal damage. We hypothesized that isoflurane will have similar effects on the polyglutamine huntingtin protein and will cause alterations in intracellular calcium homeostasis. We tested this hypothesis in striatal cells from the expanded glutamine huntingtin knock-in mouse (STHdh{sup Q111/Q111}) and wild type (STHdh{sup Q7/Q7}) striatal neurons. The primary cultured neurons were exposed for 24 h to equipotent concentrations of isoflurane, sevoflurane, and desflurane in the presence or absence of extracellular calcium and with or without xestospongin C, a potent endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP{sub 3}) receptor antagonist. Aggregation of huntingtin protein, cellmore » viability, and calcium concentrations were measured. Isoflurane, sevoflurane, and desflurane all increased the aggregation of huntingtin in STHdh{sup Q111/Q111} cells, with isoflurane having the largest effect. Isoflurane induced greater calcium release from the ER and relatively more cell damage in the STHdh{sup Q111/Q111} huntingtin cells than in the wild type STHdh{sup Q7/Q7} striatal cells. However, sevoflurane and desflurane caused less calcium release from the ER and less cell damage. Xestospongin C inhibited the isoflurane-induced calcium release from the ER, aggregation of huntingtin, and cell damage in the STHdh{sup Q111/Q111} cells. In summary, the Q111 form of huntingtin increases the vulnerability of striatal neurons to isoflurane neurotoxicity through combined actions on the ER IP{sub 3} receptors. Calcium release from the ER contributes to the anesthetic induced huntingtin aggregation in STHdh{sup Q111/Q111} striatal cells.« less

Calcium and 1,25-dihydroxyvitamin D3 modulate genes of immune and inflammatory pathways in the human colon: a human crossover trial123

PubMed Central

Protiva, Petr; Pendyala, Swaroop; Nelson, Celeste; Augenlicht, Leonard H; Lipkin, Martin; Holt, Peter R

2016-01-01

Background: A high dietary calcium intake with adequate vitamin D status has been linked to lower colorectal cancer risk, but the mechanisms of these effects are poorly understood. Objective: The objective of this study was to elucidate the effects of a Western-style diet (WD) and supplemental calcium and/or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the colorectal mucosa. Design: We conducted 2 crossover trials to define molecular pathways in the human colorectum altered by 1) a 4-wk WD supplemented with and without 2 g calcium carbonate/d and 2) a 4-wk WD supplemented with 1,25(OH)2D3 (0.5 μg/d) with or without 2 g calcium carbonate/d. The primary study endpoint was genome-wide gene expression in biopsy specimens of the rectosigmoid colonic mucosa. Serum and urinary calcium concentrations were also measured. Results: Changes in urinary calcium accurately reflected calcium consumption. The WD induced modest upregulation of genes involved in inflammatory pathways, including interferon signaling, and calcium supplementation reversed these toward baseline. In contrast, supplementation of the WD with 1,25(OH)2D3 induced striking upregulation of genes involved in inflammation, immune response, extracellular matrix, and cell adhesion. Calcium supplementation largely abrogated these changes. Conclusions: Supplementing 1,25(OH)2D3 to a WD markedly upregulated genes in immune response and inflammation pathways, which were largely reversed by calcium supplementation. This study provides clinical trial evidence of global gene expression changes occurring in the human colorectum in response to calcium and 1,25(OH)2D3 intervention. One action of 1,25(OH)2D3 is to upregulate adaptive immunity. Calcium appears to modulate this effect, pointing to its biological interaction in the mucosa. This trial was registered at clinicaltrials.gov as NCT00298545. Trial protocol is available at http://clinicalstudies.rucares.org (protocol numbers PHO475 and PHO554). PMID:27009752

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore.

PubMed

Elustondo, Pia A; Negoda, Alexander; Kane, Constance L; Kane, Daniel A; Pavlov, Evgeny V

2015-02-01

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix. Copyright © 2014 Elsevier B.V. All rights reserved.

Calcitonin control of calcium metabolism during weightlessness

NASA Technical Reports Server (NTRS)

Soliman, Karam F. A.

1993-01-01

The main objective of this proposal is to elucidate calcitonin role in calcium homeostasis during weightlessness. In this investigation our objectives are to study: the effect of weightlessness on thyroid and serum calcitonin, the effect of weightlessness on the circadian variation of calcitonin in serum and the thyroid gland, the role of light as zeitgeber for calcitonin circadian rhythm, the circadian pattern of thyroid sensitivity to release calcitonin in response to calcium load, and the role of serotonin and norepinephrine in the control of calcitonin release. The main objective of this research/proposal is to establish the role of calcitonin in calcium metabolism during weightlessness condition. Understanding the mechanism of these abnormalities will help in developing therapeutic means to counter calcium imbalance in spaceflights.

"An estimate of the probability of vesicle exocytosis in a Monte Carlo model of buffered diffusion of calcium channel currents"

NASA Astrophysics Data System (ADS)

Dimcovic, Z. M.; Eagan, T. P.; Kidane, T. K.; Brown, R. W.; Petschek, R. G.; McEnery, M. W.

2001-10-01

The opening of voltage-dependent calcium channels results in an influx of calcium ions promoting the fusion of synaptic vesicles. The fusion leads to release of neurotransmitters, which in turn allow the propagation of nerve impulses. A Monte Carlo model of the diffusion of calcium following its surge into the cell is used to estimate the probability for exocytosis. Besides the calcium absorption by fixed and mobile buffers, key ingredients are the physical size and position of the tethered vesicle and a sensing model for the interaction of the vesicle and calcium. The release probability is compared to previously published studies where the finite vesicle size was not considered. (Supported by NIH MH55747, AHA 96001250, NSF0086643, and a CWRU Presidential Research Initiative grant.)

Physicochemical properties of calcium silicate-based formulations MTA Repair HP and MTA Vitalcem

PubMed Central

Guimarães, Bruno Martini; Prati, Carlo; Duarte, Marco Antonio Hungaro; Bramante, Clovis Monteiro; Gandolfi, Maria Giovanna

2018-01-01

Abstract Objective This study aimed to analyze the following physicochemical properties: radiopacity, final setting time, calcium release, pH change, solubility, water sorption, porosity, surface morphology, and apatite-forming ability of two calcium silicate-based materials. Material and methods We tested MTA Repair HP and MTA Vitalcem in comparison with conventional MTA, analyzing radiopacity and final setting time. Water absorption, interconnected pores and apparent porosity were measured after 24-h immersion in deionized water at 37°C. Calcium and pH were tested up to 28 d in deionized water. We analyzed data using two-way ANOVA with Student-Newman-Keuls tests (p<0.05). We performed morphological and chemical analyses of the material surfaces using ESEM/EDX after 28 d in HBSS. Results MTA Repair HP showed similar radiopacity to that of conventional MTA. All materials showed a marked alkalinizing activity within 3 h, which continued for 28 d. MTA Repair HP showed the highest calcium release at 28 d (p<0.05). MTA Vitalcem showed statistically higher water sorption and solubility values (p<0.05). All materials showed the ability to nucleate calcium phosphate on their surface after 28 d in HBSS. Conclusions MTA Repair HP and MTA Vitalcem had extended alkalinizing activity and calcium release that favored calcium phosphate nucleation. The presence of the plasticizer in MTA HP might increase its solubility and porosity. The radiopacifier calcium tungstate can be used to replace bismuth oxide. PMID:29641748

Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells.

PubMed

Konermann, A; Staubwasser, M; Dirk, C; Keilig, L; Bourauel, C; Götz, W; Jäger, A; Reichert, C

2014-04-01

The aim of this study was to determine the material composition and cell-mediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure. Copyright © 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Dexamethasone increases expression of 5-lipoxygenase and its activating protein in human monocytes and THP-1 cells.

PubMed

Riddick, C A; Ring, W L; Baker, J R; Hodulik, C R; Bigby, T D

1997-05-15

The aim of this study was to assess the effect of dexamethasone on 5-lipoxygenase pathway expression in human peripheral blood monocytes and the acute monocytic leukemia cell line, THP-1. Cells were conditioned over a period of days with dexamethasone, at concentrations relevant in vivo, to study the effect of the glucocorticoid on calcium-ionophore-stimulated 5-lipoxygenase product and arachidonic acid release. The effect of dexamethasone on levels of immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and its activating protein (5-LAP) was also assessed. Dexamethasone increased the stimulated release of 5-lipoxygenase products from both monocytes and THP-1 cells in a dose-dependent fashion. The increase in product generation was not due to changes in the availability of arachidonic acid. However, immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP were increased by conditioning with dexamethasone. There was no apparent effect of the glucocorticoid on LTA4-hydrolase-immunoreactive protein levels or specific activity. We conclude that dexamethasone increases 5-lipoxygenase pathway expression in both monocytes and in THP-1 cells. This effect is due, at least in part, to increases in immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP. These results suggest a role for glucocorticoids in the regulation of 5-lipoxygenase pathway expression in mononuclear phagocytes.

Rechargeable calcium phosphate orthodontic cement with sustained ion release and re-release

PubMed Central

Zhang, Ling; Weir, Michael D.; Chow, Laurence C.; Reynolds, Mark A.; Xu, Hockin H. K.

2016-01-01

White spot lesions (WSL) due to enamel demineralization are major complications for orthodontic treatments. Calcium phosphate (CaP) dental resins with Ca and P ion releases are promising for remineralization. However, previous Ca and P releases lasted for only weeks. Experimental orthodontic cements were developed using pyromellitic glycerol dimethacrylate (PMGDM) and ethoxylated bisphenol A dimethacrylate (EBPADMA) at mass ratio of 1:1 (PE); and PE plus 10% of 2-hydroxyethyl methacrylate (HEMA) and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (PEHB). Particles of amorphous calcium phosphate (ACP) were incorporated into PE and PEHB at 40% filler level. Specimens were tested for bracket-enamel shear bond strength, water sorption, CaP release, and ion recharge and re-release. PEHB+40ACP had higher bracket-enamel bond strength and ion release and rechargeability than PE+40ACP. ACP incorporation into the novel orthodontic cement did not adversely affect the bracket-enamel bond strength. Ion release and re-release from the novel ACP orthodontic cement indicated favorable release and re-release patterns. The recharged orthodontic cement could release CaP ions continuously for four weeks without further recharge. Novel rechargeable orthodontic cement containing ACP was developed with a high bracket-enamel bond strength and the ability to be repeatedly recharged to maintain long-term high levels of CaP ion releases. PMID:27808251

TRPV1 Channels Are Functionally Coupled with BK(mSlo1) Channels in Rat Dorsal Root Ganglion (DRG) Neurons

PubMed Central

Yan, Zonghe; Kong, Wenjuan; Liu, Beiying; Li, Xia; Yao, Jing; Zhang, Yuexuan; Qin, Feng; Ding, Jiuping

2013-01-01

The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca2+). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca2+ influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the “pain” signal transduction pathway in the peripheral nervous system. PMID:24147119

Intermediate progenitors are increased by lengthening of the cell cycle through calcium signaling and p53 expression in human neural progenitors

PubMed Central

García-García, Elisa; Pino-Barrio, María José; López-Medina, Laura; Martínez-Serrano, Alberto

2012-01-01

During development, neurons can be generated directly from a multipotent progenitor or indirectly through an intermediate progenitor (IP). This last mode of division amplifies the progeny of neurons. The mechanisms governing the generation and behavior of IPs are not well understood. In this work, we demonstrate that the lengthening of the cell cycle enhances the generation of neurons in a human neural progenitor cell system in vitro and also the generation and expansion of IPs. These IPs are insulinoma-associated 1 (Insm1)+/BTG family member 2 (Btg2)−, which suggests an increase in a self-amplifying IP population. Later the cultures express neurogenin 2 (Ngn2) and become neurogenic. The signaling responsible for this cell cycle modulation is investigated. It is found that the release of calcium from the endoplasmic reticulum to the cytosol in response to B cell lymphoma-extra large overexpression or ATP addition lengths the cell cycle and increases the number of IPs and, in turn, the final neuron outcome. Moreover, data suggest that the p53–p21 pathway is responsible for the changes in cell cycle. In agreement with this, increased p53 levels are necessary for a calcium-induced increase in neurons. Our findings contribute to understand how calcium signaling can modulate cell cycle length during neurogenesis. PMID:22323293

Filamin and Phospholipase C-ε Are Required for Calcium Signaling in the Caenorhabditis elegans Spermatheca

PubMed Central

Kovacevic, Ismar; Orozco, Jose M.; Cram, Erin J.

2013-01-01

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. PMID:23671426

Uropathogenic E. coli Induce Different Immune Response in Testicular and Peritoneal Macrophages: Implications for Testicular Immune Privilege

PubMed Central

Bhushan, Sudhanshu; Hossain, Hamid; Lu, Yongning; Geisler, Andreas; Tchatalbachev, Svetlin; Mikulski, Zbigniew; Schuler, Gerhard; Klug, Jörg; Pilatz, Adrian; Wagenlehner, Florian; Chakraborty, Trinad; Meinhardt, Andreas

2011-01-01

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells. PMID:22164293

Resistance of extraocular motoneuron terminals to effects of amyotrophic lateral sclerosis sera

NASA Technical Reports Server (NTRS)

Mosier, D. R.; Siklos, L.; Appel, S. H.

2000-01-01

In sporadic ALS (s-ALS), axon terminals contain increased intracellular calcium. Passively transferred sera from patients with s-ALS increase intracellular calcium in spinal motoneuron terminals in vivo and enhance spontaneous transmitter release, a calcium-dependent process. In this study, passive transfer of s-ALS sera increased spontaneous release from spinal but not extraocular motoneuron terminals, suggesting that the resistance to physiologic abnormalities induced by s-ALS sera in mice parallels the resistance of extraocular motoneurons to dysfunction and degeneration in ALS.

Simvastatin Potently Induces Calcium-dependent Apoptosis of Human Leiomyoma Cells*

PubMed Central

Borahay, Mostafa A.; Kilic, Gokhan S.; Yallampalli, Chandrasekha; Snyder, Russell R.; Hankins, Gary D. V.; Al-Hendy, Ayman; Boehning, Darren

2014-01-01

Statins are drugs commonly used for the treatment of high plasma cholesterol levels. Beyond these well known lipid-lowering properties, they possess broad-reaching effects in vivo, including antitumor effects. Statins inhibit the growth of multiple tumors. However, the mechanisms remain incompletely understood. Here we show that simvastatin inhibits the proliferation of human leiomyoma cells. This was associated with decreased mitogen-activated protein kinase signaling and multiple changes in cell cycle progression. Simvastatin potently stimulated leiomyoma cell apoptosis in a manner mechanistically dependent upon apoptotic calcium release from voltage-gated calcium channels. Therefore, simvastatin possesses antitumor effects that are dependent upon the apoptotic calcium release machinery. PMID:25359773

Aspects of Solvent Chemistry for Calcium Hydroxide Medicaments

PubMed Central

Athanassiadis, Basil

2017-01-01

Calcium hydroxide pastes have been used in endodontics since 1947. Most current calcium hydroxide endodontic pastes use water as the vehicle, which limits the dissolution of calcium hydroxide that can be achieved and, thereby, the maximum pH that can be achieved within the root canal system. Using polyethylene glycol as a solvent, rather than water, can achieve an increase in hydroxyl ions release compared to water or saline. By adopting non-aqueous solvents such as the polyethylene glycols (PEG), greater dissolution and faster hydroxyl ion release can be achieved, leading to enhanced antimicrobial actions, and other improvements in performance and biocompatibility. PMID:29065542

Importance of vesicle release stochasticity in neuro-spike communication.

PubMed

Ramezani, Hamideh; Akan, Ozgur B

2017-07-01

Aim of this paper is proposing a stochastic model for vesicle release process, a part of neuro-spike communication. Hence, we study biological events occurring in this process and use microphysiological simulations to observe functionality of these events. Since the most important source of variability in vesicle release probability is opening of voltage dependent calcium channels (VDCCs) followed by influx of calcium ions through these channels, we propose a stochastic model for this event, while using a deterministic model for other variability sources. To capture the stochasticity of calcium influx to pre-synaptic neuron in our model, we study its statistics and find that it can be modeled by a distribution defined based on Normal and Logistic distributions.

Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways.

PubMed

Virgilio, Stela; Cupertino, Fernanda Barbosa; Ambrosio, Daniela Luz; Bertolini, Maria Célia

2017-06-09

Glycogen and trehalose are storage carbohydrates and their levels in microorganisms vary according to environmental conditions. In Neurospora crassa, alkaline pH stress highly influences glycogen levels, and in Saccharomyces cerevisiae, the response to pH stress also involves the calcineurin signaling pathway mediated by the Crz1 transcription factor. Recently, in yeast, pH stress response genes were identified as targets of Crz1 including genes involved in glycogen and trehalose metabolism. In this work, we present evidence that in N. crassa the glycogen and trehalose metabolism is modulated by alkaline pH and calcium stresses. We demonstrated that the pH signaling pathway in N. crassa controls the accumulation of the reserve carbohydrates glycogen and trehalose via the PAC-3 transcription factor, which is the central regulator of the signaling pathway. The protein binds to the promoters of most of the genes encoding enzymes of glycogen and trehalose metabolism and regulates their expression. We also demonstrated that the reserve carbohydrate levels and gene expression are both modulated under calcium stress and that the response to calcium stress may involve the concerted action of PAC-3. Calcium activates growth of the Δpac-3 strain and influences its glycogen and trehalose accumulation. In addition, calcium stress differently regulates glycogen and trehalose metabolism in the mutant strain compared to the wild-type strain. While glycogen levels are decreased in both strains, the trehalose levels are significantly increased in the wild-type strain and not affected by calcium in the mutant strain when compared to mycelium not exposed to calcium. We previously reported the role of PAC-3 as a transcription factor involved in glycogen metabolism regulation by controlling the expression of the gsn gene, which encodes an enzyme of glycogen synthesis. In this work, we extended the investigation by studying in greater detail the effects of pH on the metabolism of the reserve carbohydrate glycogen and trehalose. We also demonstrated that calcium stress affects the reserve carbohydrate levels and the response to calcium stress may require PAC-3. Considering that the reserve carbohydrate metabolism may be subjected to different signaling pathways control, our data contribute to the understanding of the N. crassa responses under pH and calcium stresses.

Sodium lauryl sulfate impedes drug release from zinc-crosslinked alginate beads: switching from enteric coating release into biphasic profiles.

PubMed

Taha, Mutasem O; Nasser, Wissam; Ardakani, Adel; Alkhatib, Hatim S

2008-02-28

The aim of this research is to investigate the effects of sodium lauryl sulfate (SLS) on ionotropically cross-linked alginate beads. Different levels of SLS were mixed with sodium alginate and chlorpheniramine maleate (as loaded model drug). The resulting viscous solutions were dropped onto aqueous solutions of zinc or calcium ions for ionotropic curing. The generated beads were assessed by their drug releasing profiles, infrared and differential scanning colorimetery (DSC) traits. SLS was found to exert profound concentration-dependent impacts on the characteristics of zinc-crosslinked alginate beads such that moderate modifications in the levels of SLS switched drug release from enteric coating-like behavior to a biphasic release modifiable to sustained-release by the addition of minute amounts of xanthan gum. Calcium cross-linking failed to reproduce the same behavior, probably due to the mainly ionic nature of calcium-carboxylate bonds compared to the coordinate character of their zinc-carboxylate counterparts. Apparently, moderate levels of SLS repel water penetration into the beads, and therefore minimize chlorpheniramine release. However, higher SLS levels seem to discourage polymeric cross-linking and therefore allow biphasic drug release.

Calcium permeable AMPA receptors and autoreceptors in external tufted cells of rat olfactory bulb

PubMed Central

Ma, Jie; Lowe, Graeme

2007-01-01

Glomeruli are functional units of the olfactory bulb responsible for early processing of odor information encoded by single olfactory receptor genes. Glomerular neural circuitry includes numerous external tufted (ET) cells whose rhythmic burst firing may mediate synchronization of bulbar activity with the inhalation cycle. Bursting is entrained by glutamatergic input from olfactory nerve terminals, so specific properties of ionotropic glutamate receptors on ET cells are likely to be important determinants of olfactory processing. Particularly intriguing is recent evidence that α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of juxta-glomerular neurons may permeate calcium. This could provide a novel pathway for regulating ET cell signaling. We tested the hypothesis that ET cells express functional calcium-permeable AMPA receptors. In rat olfactory bulb slices, excitatory postsynaptic currents (EPSCs) in ET cells were evoked by olfactory nerve shock, and by uncaging glutamate. We found attenuation of AMPA/kainate EPSCs by 1-naphthyl acetyl-spermine (NAS), an open-channel blocker specific for calcium permeable AMPA receptors. Cyclothiazide strongly potentiated EPSCs, indicating a major contribution from AMPA receptors. The current-voltage (I-V) relation of uncaging EPSCs showed weak inward rectification which was lost after > ~ 10 min of whole-cell dialysis, and was absent in NAS. In kainate-stimulated slices, Co2+ ions permeated cells of the glomerular layer. Large AMPA EPSCs were accompanied by fluorescence signals in fluo-4 loaded cells, suggesting calcium permeation. Depolarizing pulses evoked slow tail currents with pharmacology consistent with involvement of calcium permeable AMPA autoreceptors. Tail currents were abolished by Cd2+ and NBQX, and were sensitive to NAS block. Glutamate autoreceptors were confirmed by uncaging intracellular calcium to evoke a large inward current. Our results provide evidence that calcium permeable AMPA receptors reside on ET cells, and are divided into at least two functionally distinct pools – postsynaptic receptors at olfactory nerve synaptic terminals, and autoreceptors sensitive to glutamate released from dendrodendritic synapses. PMID:17156930

Influence of 2% chlorhexidine on pH, calcium release and setting time of a resinous MTA-based root-end filling material.

PubMed

Jacinto, Rogério Castilho; Linhares-Farina, Giane; Sposito, Otávio da Silva; Zanchi, César Henrique; Cenci, Maximiliano Sérgio

2015-01-01

The addition of chlorhexidine (CHX) to a resinous experimental Mineral Trioxide Aggregate (E-MTA) based root-end filling material is an alternative to boost its antimicrobial activity. However, the influence of chlorhexidine on the properties of this material is unclear. The aim of this study was to evaluate the influence of 2% chlorhexidine on the pH, calcium ion release and setting time of a Bisphenol A Ethoxylate Dimethacrylate/Mineral Trioxide Aggregate (Bis-EMA/MTA) based dual-cure experimental root-end filling material (E-MTA), in comparison with E-MTA without the addition of CHX and with conventional white MTA (W-MTA). The materials were placed in polyethylene tubes, and immersed in deionized water to determine pH (digital pH meter) and calcium ion release (atomic absorption spectrometry technique). The setting time of each material was analyzed using Gilmore needles. The data were statistically analyzed at a significance level of 5%. E-MTA + CHX showed an alkaline pH in the 3 h period of evaluation, the alkalinity of which decreased but remained as such for 15 days. The pH of E-MTA + CHX was higher than the other two materials after 7 days, and lower after 30 days (p < 0.05). All of the materials were found to release calcium ions throughout the 30 days of the study. The addition of CHX increased the calcium ion release of E-MTA to levels statistically similar to W-MTA. E-MTA showed shorter initial and final setting time, compared with W-MTA (p < 0.05). The addition of 2% CHX to MTA prevented setting of the material. The addition of CHX to E-MTA increased its pH and calcium ion release. However, it also prevented setting of the material.

Control of arachidonic acid release in chick muscle cultures

NASA Technical Reports Server (NTRS)

Templeton, G. H.; Padalino, M.; Wright, W.

1985-01-01

Cultures from thigh muscles of 12 day old embryonic chicks are utilized to examine arachidonic release, prostaglandin (PG) biosynthesis, and protein synthesis. The preparation of the cultures is described. It is observed that exogenous arachidonic acid is formed into photsphatidylethanolamine and phosphatidylcholine, is released by a calcium ionosphere or phospholiphase simulator, and is the substrate for the biosynthesis of PG; the epidermal growth factor and PGF do not stimulate protein synthesis over the basal levels. The relationship between arachidonate release and melittin is studied. The data reveal that a change in intracellular calcium stimulates phospholiphase activity, arachidonate release, and PG synthesis in chick muscle culture.

[The alpha2delta subunit of the voltage-dependent calcium channel. A new pharmaceutical target for psychiatry and neurology].

PubMed

Wedekind, D; Bandelow, B

2005-07-01

Calcium channel blockers are substances used for treating high blood pressure and coronary heart disease. New medications have been developed that modulate calcium channels but also show promise in psychiatric and neurologic applications. Gabapentin and pregabalin bind to a subunit of calcium channels--the alpha2delta receptors--thereby reducing calcium influx to neurons. As a result, less glutamate is released from nerve endings that use excitatory amino acids as transmitters. This in turn reduces substance P-related activation of AMPA heteroreceptors on noradrenergic synapses, total transmitter release, and finally neuronal activity. That mechanism is the probable explanation for gabapentin's and pregabalin's usefulness in the treatment of neuropathic pain but also their possible anticonvulsive and anxiolytic effects.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty

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 bindingmore » 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.« less

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

PubMed Central

Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

2017-01-01

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission. PMID:28241053

Calcium silicate-based sealers: Assessment of physicochemical properties, porosity and hydration.

PubMed

Marciano, Marina Angélica; Duarte, Marco Antonio Hungaro; Camilleri, Josette

2016-02-01

Investigation of hydration, chemical, physical properties and porosity of experimental calcium silicate-based sealers. Experimental calcium silicate-based sealers with calcium tungstate and zirconium oxide radio-opacifiers were prepared by mixing 1g of powder to 0.3 mL of 80% distilled water and 20% propylene glycol. MTA and MTA Fillapex were used as controls. The raw materials and set sealers were characterized using a combination of scanning electron microscopy, energy dispersive spectroscopy and X-ray diffraction. Physical properties were analyzed according to ANSI/ADA. The pH and calcium ion release were assessed after 3, 24, 72 and 168 h. The porosity was assessed using mercury intrusion porosimetry. The analysis of hydration of prototype sealers revealed calcium hydroxide as a by-product resulting in alkaline pH and detection of calcium ion release, with high values in initial periods. The radiopacity was similar to MTA for the sealers containing high amounts of radio-opacifiers (p>0.05). Flowability was higher and film thickness was lower for resinous MTA Fillapex sealer (p<0.05). The test sealers showed water sorption and porosity similar to MTA (p>0.05). The prototype sealers presented adequate hydration, elevated pH and calcium ion release. Regarding physical properties, elevated proportions of radio-opacifiers were necessary to accomplish adequate radiopacity, enhance flowability and reduce film thickness. All the tested sealers presented water sorption and porosity similar to MTA. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

A Mechanism of Intracellular P2X Receptor Activation*

PubMed Central

Sivaramakrishnan, Venketesh; Fountain, Samuel J.

2012-01-01

P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2XAR knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen. PMID:22736763

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development.

PubMed

Webb, Sarah E; Miller, Andrew L

2006-11-01

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming pathways known to modulate early developmental events (such as the Wnt/Ca(2+)pathway; [T.A. Westfall, B. Hjertos, D.C. Slusarski, Requirement for intracellular calcium modulation in zebrafish dorsal-ventral patterning, Dev. Biol. 259 (2003) 380-391]).

Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations.

PubMed

Rice, Louise V; Bax, Heather J; Russell, Linda J; Barrett, Victoria J; Walton, Sarah E; Deakin, Angela M; Thomson, Sally A; Lucas, Fiona; Solari, Roberto; House, David; Begg, Malcolm

2013-03-15

Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Transgenic Analysis of the Role of FKBP12.6 in Cardiac Function and Intracellular Calcium Release

PubMed Central

Liu, Ying; Chen, Hanying; Ji, Guangju; Li, Baiyan; Mohler, Peter J.; Zhu, Zhiming; Yong, Weidong; Chen, Zhuang; Xu, Xuehong

2011-01-01

Abstract FK506 binding protein12.6 (FKBP12.6) binds to the Ca2+ release channel ryanodine receptor (RyR2) in cardiomyocytes and stabilizes RyR2 to prevent premature sarcoplasmic reticulum Ca2+ release. Previously, two different mouse strains deficient in FKBP12.6 were reported to have different abnormal cardiac phenotypes. The first mutant strain displayed sex-dependent cardiac hypertrophy, while the second displayed exercise-induced cardiac arrhythmia and sudden death. In this study, we tested whether FKBP12.6-deficient mice that display hypertrophic hearts can develop exercise-induced cardiac sudden death and whether the hypertrophic heart is a direct consequence of abnormal calcium handling in mutant cardiomyocytes. Our data show that FKBP12.6-deficient mice with cardiac hypertrophy do not display exercise-induced arrhythmia and/or sudden cardiac death. To investigate the role of FKBP12.6 overexpression for cardiac function and cardiomyocyte calcium release, we generated a transgenic mouse line with cardiac specific overexpression of FKBP12.6 using α-myosin heavy chain (αMHC) promoter. MHC-FKBP12.6 mice displayed normal cardiac development and function. We demonstrated that MHC-FKBP12.6 mice are able to rescue abnormal cardiac hypertrophy and abnormal calcium release in FKBP12.6-deficient mice. PMID:22087651

Electrosynthesis of magnetoresponsive microrobot for targeted drug delivery using calcium alginate.

PubMed

Chengzhi Hu; Riederer, Katharina; Klemmer, Michael; Pane, Salvador; Nelson, Bradley J

2016-08-01

Targeted drug delivery systems deliver drugs precisely to a specific targeted site inside the body, and can also release the drugs with controlled kinetics to prolong the efficacy of single dose administration. The advantageous properties of hydrogels make them attractive for use in the area of drug delivery. Calcium alginate is a pH sensitive hydrogel stable in acidic media and soluble in basic media. This enables the hydrogel to absorb and release aqueous solutions at certain ranges of pH values. By absorbing an aqueous solution containing a drug, an active drug release can be triggered at a specified range of pH value. In this paper, we combined calcium alginate with cobalt nickel (CoNi) in a cylindrical hybrid micro robot by electrodeposition. The designed microrobot can be wirelessly actuated with an external magnetic manipulation system and, hence, targeted to a specific location in the human body. At this specific location, characterized by its pH range, the absorbed drug will be released. Here, the fabrication steps of the specified microrobot are characterized, namely the production of a template on a silicon chip and the subsequent template-assisted electrodeposition of CoNi and alginate. Additionally, the dynamics of drug release of calcium alginate is studied.

Thermal Decomposition of Calcium Perchlorate/Iron-Mineral Mixtures: Implications of the Evolved Oxygen from the Rocknest Eolian Deposit in Gale Crater, Mars

NASA Technical Reports Server (NTRS)

Bruck, A. M.; Sutter, B.; Ming, D. W.; Mahaffy, P.

2014-01-01

A major oxygen release between 300 and 500 C was detected by the Mars Curiosity Rover Sample Analysis at Mars (SAM) instrument at the Rocknest eolian deposit. Thermal decomposition of perchlorate (ClO4-) salts in the Rocknest samples are a possible explanation for this evolved oxygen release. Releative to Na-, K-, Mg-, and Fe-perchlorate, the thermal decomposition of Ca-perchlorate in laboratory experiments released O2 in the temperature range (400-500degC) closest to the O2 release temperatures observed for the Rocknest material. Furthermore, calcium perchlorate could have been the source of Cl in the chlorinated-hydrocarbons species that were detected by SAM. Different components in the Martian soil could affect the decomposition temperature of calcium per-chlorate or another oxychlorine species. This interaction of the two components in the soil could result in O2 release temperatures consistent with those detected by SAM in the Rocknest materials. The decomposition temperatures of various alkali metal perchlorates are known to decrease in the presence of a catalyst. The objective of this work is to investigate catalytic interactions on calcium perchlorate from various iron-bearing minerals known to be present in the Rocknest material

Endocrine and metabolic changes in transition dairy cows are affected by prepartum infusions of a serotonin precursor.

PubMed

Hernández-Castellano, Lorenzo E; Hernandez, Laura L; Sauerwein, Helga; Bruckmaier, Rupert M

2017-06-01

Serotonin (5-HT) has been shown to be involved in calcium homeostasis, modulating calcium concentration in blood. In addition, 5-HT participates in a variety of metabolic pathways, mainly through the modulation of glucose and lipid metabolism. The hypothesis of the present study was that the prepartum administration of 5-hydroxy-l-tryptophan (5-HTP), a 5-HT precursor, would affect endocrine systems related to calcium homeostasis, and interact with other endocrine and metabolic pathways during the transition period. In this study, 20 Holstein dairy cows were randomly assigned to 2 experimental groups. Both groups received a daily i.v. infusion of 1 L of either 0.9% NaCl (control group; n = 10) or 0.9% NaCl containing 1 mg of 5-HTP/kg of BW (5-HTP group, n = 10). Infusions started d 10 before estimated parturition date and ended the day of parturition, resulting in a minimum of 4 d of infusion (8.4 ± 0.7 d of infusion). Until parturition, blood samples were collected before the daily infusions, and postpartum daily until d 7, and on d 30. Plasma concentrations of parathyroid hormone (PTH) were transiently increased at parturition and on d 1 in control cows. In the 5-HTP group PTH remained unchanged. The concentration of pyridinoline (PYD), an established marker for calcium release from the bone to the bloodstream, increased on d 1 postpartum only in the 5-HTP group. In control cows, PYD concentrations did not change on d 1 postpartum. Melatonin concentrations were slightly but significantly increased in the 5-HTP group compared with the control group. Insulin concentrations decreased in both groups postpartum. Before parturition, leptin concentrations decreased in both groups and remained at this level until d 30 postpartum. Plasma IgG concentrations decreased in both groups on d -1 postpartum. Haptoglobin increased in both groups on d -1 and remained at this level until d 7 postpartum. No differences between groups were observed for insulin, glucagon, IgG, leptin, adiponectin, and haptoglobin concentrations. The results obtained in the present study evidenced that 5-HT is regulating calcium homeostasis independent of PTH. The lack of treatment effects on IgG and on other hormones and metabolites indicates that 5-HTP did not affect these other metabolic pathways and the IgG concentration during the transition period. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, E.L.; Singh, J.C.; Jacobson, K.L.

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated /sup 45/Ca/sup 2 +/ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10/sup -8/M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 x 10/sup -7/more » M) or hydroxylamine (50 ..mu..M) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism. 21 references, 1 figure, 3 tables.« less

Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters

ERIC Educational Resources Information Center

Nicholl, Peter A.; Howlett, Susan E.

2006-01-01

Whether the density of sarcoplasmic reticulum (SR) calcium release channels/ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of [3H]-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes.…

40 CFR 721.10599 - Calcium cobalt lead titanium tungsten oxide.

Code of Federal Regulations, 2013 CFR

2013-07-01

... systems). (iii) Release to water. Requirements as specified in § 721.90 (a)(4), (b)(4), and (c)(4) (Where N=8, and 8 is an aggregate of releases for the following substances: Lead strontium titanium...-271; CAS No. 1262279-31-1); Calcium cobalt lead strontium titanium tungsten oxide (PMN P-11-272; CAS...

40 CFR 721.10599 - Calcium cobalt lead titanium tungsten oxide.

Code of Federal Regulations, 2014 CFR

2014-07-01

... systems). (iii) Release to water. Requirements as specified in § 721.90 (a)(4), (b)(4), and (c)(4) (Where N=8, and 8 is an aggregate of releases for the following substances: Lead strontium titanium...-271; CAS No. 1262279-31-1); Calcium cobalt lead strontium titanium tungsten oxide (PMN P-11-272; CAS...

Transmitter release and presynaptic Ca2+ currents blocked by the spider toxin omega-Aga-IVA.

PubMed

Protti, D A; Uchitel, O D

1993-12-13

Mammalian neuromuscular transmission is resistant to L and N type calcium channel blockers but very sensitive to a low molecular weight funnel web spider venom toxin, FTX, which selectively blocks P type calcium channels. To further characterize the calcium channels involved in neuromuscular transmission we studied the effect of omega Agatoxin (omega-Aga-IVA) a polypeptide P type channel blocker from the same spider venom. We show that omega-Aga-IVA is a potent and irreversible inhibitor of the presynaptic Ca2+ currents and of acetylcholine release induced by electrical stimulation or by K+ depolarization. This provides further evidences that transmitter release at the mammalian neuromuscular junction is mediated by P type Ca2+ channels.

Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture.

PubMed

Belemtougri, R G; Constantin, B; Cognard, C; Raymond, G; Sawadogo, L

2006-01-01

Crude decoction, aqueous and ethanolic extracts of two medicinal plants (Psidium guajava and Diospyros mespiliformis), widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidium guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explain their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release.

Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture

PubMed Central

Belemtougri, R.G.; Constantin, B.; Cognard, C.; Raymond, G.; Sawadogo, L.

2006-01-01

Crude decoction, aqueous and ethanolic extracts of two medicinal plants (Psidium guajava and Diospyros mespiliformis), widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidium guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explaine their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release. PMID:16365927

Physiological and anatomical studies of the development of the sympathetic innervation to rat iris arterioles.

PubMed

Sandow; Hill

1999-09-24

The development of the sympathetic innervation to rat irideal arterioles has been investigated using histochemical and in vitro pharmacological and electrophysiological methods. A plexus of fibres and varicosities appeared over the surface of the vessels after the first postnatal week and increased to reach a maximum density during the fourth postnatal week. Transmural nerve stimulation produced small, consistent contractions that were first recorded in arterioles of 7-day old rats. Contractions became larger and faster, reaching the adult form during the fourth postnatal week. Contractions became more sensitive to the alpha1-adrenoceptor antagonists, prazosin and naftopidil, and less sensitive to the alpha1A/D antagonist, WB4101 and alpha2 antagonist, yohimbine, during development. At both 10 and 21 days, contractile responses resulted from the release of intracellular calcium as they were abolished by caffeine (10(-3) M), thapsigargin (2 x 10(-6) M) and cyclopiazonic acid (3 x 10(-6) M), but not by nifedipine (10(-6) M). Intracellular recordings showed that nerve stimulation produced large, slow depolarizations at all ages tested. Time to peak potential decreased during development, while the amplitude of the depolarizations did not vary significantly. Results suggest that, throughout development, sympathetic nerves cause constriction of iris arterioles due to the release of noradrenaline and activation of alpha-adrenoceptors on the smooth muscle cells. Early responses involved both alpha1- and alpha2-adrenoceptors, while later responses were due to alpha1-adrenoceptors only. Irrespective of these changes in adrenoceptor subtypes, smooth muscle contraction resulted from the mobilization of intracellular calcium suggesting that both alpha1- and alpha2-adrenoceptors were coupled to pathways which accessed this source of calcium.

Diadenosine tetraphosphate (Ap4A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

PubMed

Reigada, David; Navarro-Ruiz, Rosa María; Caballero-López, Marcos Javier; Del Águila, Ángela; Muñoz-Galdeano, Teresa; Maza, Rodrigo M; Nieto-Díaz, Manuel

2017-03-01

Reducing cell death during the secondary injury is a major priority in the development of a cure for traumatic spinal cord injury (SCI). One of the earliest processes that follow SCI is the excitotoxicity resulting from the massive release of excitotoxicity mediators, including ATP, which induce an excessive and/or prolonged activation of their receptors and a deregulation of the calcium homeostasis. Diadenosine tetraphosphate (Ap 4 A) is an endogenous purinergic agonist, present in both extracellular and intracellular fluids, with promising cytoprotective effects in different diseases including neurodegenerative processes. In a search for efficient neuroprotective strategies for SCI, we have tested the capability of Ap 4 A to reduce the excitotoxic death mediated by the ATP-induced deregulation of calcium homeostasis and its consequences on tissue preservation and functional recovery in a mouse model of moderate contusive SCI. Our analyses with the murine neural cell line Neuro2a demonstrate that treatment with Ap 4 A reduces ATP-dependent excitotoxic death by both lowering the intracellular calcium response and decreasing the expression of specific purinergic receptors. Follow-up analyses in a mouse model of contusive SCI showed that acute administration of Ap 4 A following SCI reduces tissue damage and improves motor function recovery. These results suggest that Ap 4 A cytoprotection results from a decrease of the purinergic tone preventing the effects of a massive release of ATP after SCI, probably together with a direct induction of anti-apoptotic and pro-survival pathways via activation of P2Y 2 proposed in previous studies. In conclusion, Ap 4 A may be a good candidate for an SCI therapy, particularly to reduce excitotoxicity in combination with other modulators and/or inhibitors of the excitotoxic process that are being tested.

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells.

PubMed

Vereb, G; Szöllösi, J; Mátyus, L; Balázs, M; Hyun, W C; Feuerstein, B G

1996-05-01

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.

Pectin gel vehicles for controlled fragrance delivery.

PubMed

Liu, LinShu; Chen, Guoying; Fishman, Marshall L; Hicks, Kevin B

2005-01-01

Using citronellal as a model compound, pectin gels formulations were evaluated for the controlled fragrance release by kinetic and static methods. The pectins with higher degrees of esterification induced a stronger molecular association with the nonpolar fragrance. This resulted in a prolonged duration of fragrance release and the limitation of fragrance adsorption to the receptor skin layers. The increase in pectin concentrations suppressed the fragrance release by a diffusion mechanism. Blocking the carboxyl groups of pectin with calcium ions reduces the hydrophilicity of pectin and provides physical barriers for citronellal diffusion. The pectin/calcium microparticles are promising materials for controlled fragrance release.

Neuroprotective Effects of Sulforaphane after Contusive Spinal Cord Injury

PubMed Central

Benedict, Andrea L.; Mountney, Andrea; Hurtado, Andres; Bryan, Kelley E.; Schnaar, Ronald L.; Dinkova-Kostova, Albena T.

2012-01-01

Abstract Traumatic spinal cord injury (SCI) leads to oxidative stress, calcium mobilization, glutamate toxicity, the release of proinflammatory factors, and depletion of reduced glutathione (GSH) at the site of injury. Induction of the Keap1/Nrf2/ARE pathway can alleviate neurotoxicity by protecting against GSH depletion, oxidation, intracellular calcium overload, mitochondrial dysfunction, and excitotoxicity. Sulforaphane (SF), an isothiocyanate derived from broccoli, is a potent naturally-occurring inducer of the Keap1/Nrf2/ARE pathway, leading to upregulation of genes encoding cytoprotective proteins such as NAD(P)H: quinone oxidoreductase 1, and GSH-regulatory enzymes. Additionally, SF can attenuate inflammation by inhibiting the nuclear factor-κB (NF-κB) pathway, and the enzymatic activity of the proinflammatory cytokine macrophage inhibitory factor (MIF). Our study examined systemic administration of SF in a rat model of contusion SCI, in an effort to utilize its indirect antioxidant and anti-inflammatory properties to decrease secondary injury. Two doses of SF (10 or 50 mg/kg) were administered at 10 min and 72 h after contusion SCI. SF (50 mg/kg) treatment resulted in both acute and long-term beneficial effects, including upregulation of the phase 2 antioxidant response at the injury site, decreased mRNA levels of inflammatory cytokines (i.e., MMP-9) in the injured spinal cord, inactivation of urinary MIF tautomerase activity, enhanced hindlimb locomotor function, and an increased number of serotonergic axons caudal to the lesion site. These findings demonstrate that SF provides neuroprotective effects in the spinal cord after injury, and could be a candidate for therapy of SCI. PMID:22853439

Neuroprotective effects of sulforaphane after contusive spinal cord injury.

PubMed

Benedict, Andrea L; Mountney, Andrea; Hurtado, Andres; Bryan, Kelley E; Schnaar, Ronald L; Dinkova-Kostova, Albena T; Talalay, Paul

2012-11-01

Traumatic spinal cord injury (SCI) leads to oxidative stress, calcium mobilization, glutamate toxicity, the release of proinflammatory factors, and depletion of reduced glutathione (GSH) at the site of injury. Induction of the Keap1/Nrf2/ARE pathway can alleviate neurotoxicity by protecting against GSH depletion, oxidation, intracellular calcium overload, mitochondrial dysfunction, and excitotoxicity. Sulforaphane (SF), an isothiocyanate derived from broccoli, is a potent naturally-occurring inducer of the Keap1/Nrf2/ARE pathway, leading to upregulation of genes encoding cytoprotective proteins such as NAD(P)H: quinone oxidoreductase 1, and GSH-regulatory enzymes. Additionally, SF can attenuate inflammation by inhibiting the nuclear factor-κB (NF-κB) pathway, and the enzymatic activity of the proinflammatory cytokine macrophage inhibitory factor (MIF). Our study examined systemic administration of SF in a rat model of contusion SCI, in an effort to utilize its indirect antioxidant and anti-inflammatory properties to decrease secondary injury. Two doses of SF (10 or 50 mg/kg) were administered at 10 min and 72 h after contusion SCI. SF (50 mg/kg) treatment resulted in both acute and long-term beneficial effects, including upregulation of the phase 2 antioxidant response at the injury site, decreased mRNA levels of inflammatory cytokines (i.e., MMP-9) in the injured spinal cord, inactivation of urinary MIF tautomerase activity, enhanced hindlimb locomotor function, and an increased number of serotonergic axons caudal to the lesion site. These findings demonstrate that SF provides neuroprotective effects in the spinal cord after injury, and could be a candidate for therapy of SCI.

Intracellular calcium movements during excitation–contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers

PubMed Central

Hollingworth, Stephen

2012-01-01

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca2+) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca2+ indicator dye reveal that the amount of Ca2+ released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca2+ that binds to troponin to activate contraction is substantially smaller. PMID:22450485

The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

USDA-ARS?s Scientific Manuscript database

Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

Two Mechanisms Involved in Trigeminal CGRP Release: Implications for Migraine Treatment

PubMed Central

Durham, Paul L.; Masterson, Caleb G.

2012-01-01

Objective The goal of this study was to better understand the cellular mechanisms involved in proton stimulation of calcitonin gene-related peptide (CGRP) secretion from cultured trigeminal neurons by investigating the effects of two anti-migraine therapies, onabotulinumtoxin A and rizatriptan. Background Stimulated CGRP release from peripheral and central terminating processes of trigeminal ganglia neurons is implicated in migraine pathology by promoting inflammation and nociception. Based on models of migraine pathology, several inflammatory molecules including protons are thought to facilitate sensitization and activation of trigeminal nociceptive neurons and stimulate CGRP secretion. Despite the reported efficacy of triptans and onabotulinumtoxinA to treat acute and chronic migraine, respectively, a substantial number of migraneurs do not get adequate relief with these therapies. A possible explanation is that triptans and onabutulinumtoxinA are not able to block proton mediated CGRP secretion. Methods CGRP secretion from cultured primary trigeminal ganglia neurons was quantitated by radioimmunoassay while intracellular calcium and sodium levels were measured in neurons via live cell imaging using Fura2-AM and SBFI-AM, respectively. The expression of ASIC3 was determined by immunocytochemistry and western blot analysis. In addition, the involvement of ASICs in mediating proton stimulation of CGRP was investigated using the potent and selective ASIC3 inhibitor APETx2. Results While KCl caused a significant increase in CGRP secretion that was significantly repressed by treatment with EGTA, onabotulinumtoxinA, and rizatriptan, the stimulatory effect of protons (pH 5.5) was not suppressed by EGTA, onabotulinumtoxinA, or rizatriptan. In addition, while KCl caused a transient increase in intracellular calcium levels that was blocked by EGTA, no appreciable change in calcium levels was observed with proton treatment. However, protons did significantly increase the intracellular level of sodium ions. Under our culture conditions, ASIC3 was shown to be expressed in most trigeminal ganglion neurons. Importantly, proton stimulation of CGRP secretion was repressed by pretreatment with the ASIC3 inhibitor APETx2, but not the TRPV1 antagonist capsazepine. Conclusions Our findings provide evidence that proton regulated release of CGRP from trigeminal neurons utilizes a different mechanism than the calcium and SNAP-25 dependent pathways that are inhibited by the anti-migraine therapies rizatriptan and onabotulinumtoxinA. PMID:23095108

Aphanomyces euteiches cell wall fractions containing novel glucan-chitosaccharides induce defense genes and nuclear calcium oscillations in the plant host Medicago truncatula.

PubMed

Nars, Amaury; Lafitte, Claude; Chabaud, Mireille; Drouillard, Sophie; Mélida, Hugo; Danoun, Saïda; Le Costaouëc, Tinaig; Rey, Thomas; Benedetti, Julie; Bulone, Vincent; Barker, David George; Bono, Jean-Jacques; Dumas, Bernard; Jacquet, Christophe; Heux, Laurent; Fliegmann, Judith; Bottin, Arnaud

2013-01-01

N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.

Aphanomyces euteiches Cell Wall Fractions Containing Novel Glucan-Chitosaccharides Induce Defense Genes and Nuclear Calcium Oscillations in the Plant Host Medicago truncatula

PubMed Central

Nars, Amaury; Lafitte, Claude; Chabaud, Mireille; Drouillard, Sophie; Mélida, Hugo; Danoun, Saïda; Le Costaouëc, Tinaig; Rey, Thomas; Benedetti, Julie; Bulone, Vincent; Barker, David George; Bono, Jean-Jacques; Dumas, Bernard; Jacquet, Christophe; Heux, Laurent; Fliegmann, Judith; Bottin, Arnaud

2013-01-01

N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes. PMID:24086432

Examination of Calcium Silicate Cements with Low-Viscosity Methyl Cellulose or Hydroxypropyl Cellulose Additive.

PubMed

Baba, Toshiaki; Tsujimoto, Yasuhisa

2016-01-01

The purpose of this study was to improve the operability of calcium silicate cements (CSCs) such as mineral trioxide aggregate (MTA) cement. The flow, working time, and setting time of CSCs with different compositions containing low-viscosity methyl cellulose (MC) or hydroxypropyl cellulose (HPC) additive were examined according to ISO 6876-2012; calcium ion release analysis was also conducted. MTA and low-heat Portland cement (LPC) including 20% fine particle zirconium oxide (ZO group), LPC including zirconium oxide and 2 wt% low-viscosity MC (MC group), and HPC (HPC group) were tested. MC and HPC groups exhibited significantly higher flow values and setting times than other groups ( p < 0.05). Additionally, flow values of these groups were higher than the ISO 6876-2012 reference values; furthermore, working times were over 10 min. Calcium ion release was retarded with ZO, MC, and HPC groups compared with MTA. The concentration of calcium ions was decreased by the addition of the MC or HPC group compared with the ZO group. When low-viscosity MC or HPC was added, the composition of CSCs changed, thus fulfilling the requirements for use as root canal sealer. Calcium ion release by CSCs was affected by changing the CSC composition via the addition of MC or HPC.

Examination of Calcium Silicate Cements with Low-Viscosity Methyl Cellulose or Hydroxypropyl Cellulose Additive

PubMed Central

Tsujimoto, Yasuhisa

2016-01-01

The purpose of this study was to improve the operability of calcium silicate cements (CSCs) such as mineral trioxide aggregate (MTA) cement. The flow, working time, and setting time of CSCs with different compositions containing low-viscosity methyl cellulose (MC) or hydroxypropyl cellulose (HPC) additive were examined according to ISO 6876-2012; calcium ion release analysis was also conducted. MTA and low-heat Portland cement (LPC) including 20% fine particle zirconium oxide (ZO group), LPC including zirconium oxide and 2 wt% low-viscosity MC (MC group), and HPC (HPC group) were tested. MC and HPC groups exhibited significantly higher flow values and setting times than other groups (p < 0.05). Additionally, flow values of these groups were higher than the ISO 6876-2012 reference values; furthermore, working times were over 10 min. Calcium ion release was retarded with ZO, MC, and HPC groups compared with MTA. The concentration of calcium ions was decreased by the addition of the MC or HPC group compared with the ZO group. When low-viscosity MC or HPC was added, the composition of CSCs changed, thus fulfilling the requirements for use as root canal sealer. Calcium ion release by CSCs was affected by changing the CSC composition via the addition of MC or HPC. PMID:27981048

Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

PubMed

Yoon, Mi Na; Kim, Min Jae; Koong, Hwa Soo; Kim, Dong Kwan; Kim, Se Hoon; Park, Hyung Seo

2017-09-01

Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100 mM reversibly suppressed CCh-induced Ca 2+ oscillation in a dose-dependent manner. Pretreatment of ethanol has no effect on the store-operated calcium entry induced by 10 μM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Yolk-Shell Porous Microspheres of Calcium Phosphate Prepared by Using Calcium L-Lactate and Adenosine 5'-Triphosphate Disodium Salt: Application in Protein/Drug Delivery.

PubMed

Ding, Guan-Jun; Zhu, Ying-Jie; Qi, Chao; Sun, Tuan-Wei; Wu, Jin; Chen, Feng

2015-06-26

A facile and environmentally friendly approach has been developed to prepare yolk-shell porous microspheres of calcium phosphate by using calcium L-lactate pentahydrate (CL) as the calcium source and adenosine 5'-triphosphate disodium salt (ATP) as the phosphate source through the microwave-assisted hydrothermal method. The effects of the concentration of CL, the microwave hydrothermal temperature, and the time on the morphology and crystal phase of the product are investigated. The possible formation mechanism of yolk-shell porous microspheres of calcium phosphate is proposed. Hemoglobin from bovine red cells (Hb) and ibuprofen (IBU) are used to explore the application potential of yolk-shell porous microspheres of calcium phosphate in protein/drug loading and delivery. The experimental results indicate that the as-prepared yolk-shell porous microspheres of calcium phosphate have relatively high protein/drug loading capacity, sustained protein/drug release, favorable pH-responsive release behavior, and a high biocompatibility in the cytotoxicity test. Therefore, the yolk-shell porous microspheres of calcium phosphate have promising applications in various biomedical fields such as protein/drug delivery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsumura, Yasuo; Kawazoe, Shinka; Ichihara, Toshio

Extracellular high potassium inhibits renin release in vitro by increasing calcium concentrations in the juxtaglomerular cells. The authors found that the decreased response of renin release from rat kidney cortical slices in high potassium solution changed to a strikingly increased one in the presence of nifedipine at doses over 10{sup {minus}6} M. They then examined the stimulatory effect of extracellular high potassium in the presence of nifedipine on renin release. The enhancement of release was significantly suppressed either by propranolol or by metoprolol but not by prazosin. High potassium plus nifedipine-induced increase in renin release was markedly attenuated by renalmore » denervation. The enhancing effect was not observed when the slices were incubated in calcium-free medium. Divalent cations such as Cd{sup 2+}, Co{sup 2+}, and Mn{sup 2+} blocked this enhancement in a concentration-dependent manner. High potassium elicited an increase in {sup 3}H efflux from the slices preloaded with ({sup 3}H)-norepinephrine. The increasing effect was not influenced by nifedipine but was abolished by the removal of extracellular calcium or by the addition of divalent cations. These observations suggest to us that the high potassium plus nifedipine-induced increase in renin release from the slices is mediated by norepinephrine derived from renal sympathetic nerves and that this neuronally released norepinephrine stimulates renin release via activation of {beta}-adrenoceptors.« less

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurokawa, Yuko; Sekiguchi, Fumiko; Kubo, Satoko

2011-11-04

Highlights: Black-Right-Pointing-Pointer Hydrogen sulfide causes NMDA receptor-independent neurotoxicity in mouse fetal cortical neurons. Black-Right-Pointing-Pointer Activation of ERK mediates the toxicity of hydrogen sulfide. Black-Right-Pointing-Pointer Apoptotic mechanisms are involved in the hydrogen-induced cell death. -- Abstract: Hydrogen sulfide (H{sub 2}S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H{sub 2}S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypanmore » blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H{sub 2}S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.« less

ERP44 inhibits human lung cancer cell migration mainly via IP3R2.

PubMed

Huang, Xue; Jin, Meng; Chen, Ying-Xiao; Wang, Jun; Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-06-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

ERP44 inhibits human lung cancer cell migration mainly via IP3R2

PubMed Central

Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-01-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. PMID:27347718

Structure and reactivity of ferrihydrite-soil organic carbon-calcium ternary complexes

NASA Astrophysics Data System (ADS)

Yang, Y.; Adhikari, D.; Sowers, T.; Stuckey, J.; Poulson, S.; Sparks, D. L.

2017-12-01

Complete understanding about the interactions between soil organic carbon (SOC) and minerals is important for predicting the stability of SOC and its response to climate change. Recent studies have shown the importance of calcium (Ca)-bearing minerals and iron (Fe) oxide in associating with and stabilizing SOC. In this study, we have investigated the formation and reactivity of ferrihydrite-SOC-Ca ternary complexes. During the co-precipitation of ferrihydrite with SOC in the presence of Ca2+, 60% of SOC can be co-precipitated with ferrihydrite at a C/Fe (molar ratio) of up to 10, whereas the Ca/Fe ratio was saturated at 0.2. Increasing amount of Ca2+ did not affect the co-precipitation of SOC with ferrihydrite or the lability of ferrihydrite-bound SOC. In addition, microbial reduction of ferrihydrite and reductive release of ferrihydrite-bound SOC were not influenced by the presence of Ca, but the pathway for Fe mineral transformation during the reduction was affected by Ca. In the meantime, Fe reduction selectively released carboxylic-enriched SOC. As a comparison, the presence of SOC increased the incorporation of Ca into the structure of ferrihydrite. Our results indicate the formation of ferrihydrite-SOC-Ca complexes, with organic carbon bridging the ferrihydrite and Ca. Such ternary complexes potentially play an important role in regulating the interactions between SOC and mineral phases in soil.

CBL-CIPK network for calcium signaling in higher plants

NASA Astrophysics Data System (ADS)

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.

Calcium-dependent phosphodiesterase 1C inhibits renin release from isolated juxtaglomerular cells

PubMed Central

Ortiz-Capisano, M. Cecilia; Liao, Tang-Dong; Ortiz, Pablo A.

2009-01-01

Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by calcium. We previously showed JG cells contain a calcium sensing receptor (CaSR), which, when stimulated, decreases cAMP formation and inhibits renin release. We hypothesize CaSR activation decreases cAMP and renin release, in part, by stimulating a calcium calmodulin-activated phosphodiesterase 1 (PDE1). We incubated our primary culture of JG cells with two selective PDE1 inhibitors [8-methoxymethil-IBMX (8-MM-IBMX; 20 μM) and vinpocetine (40 μM)] and the calmodulin inhibitor W-7 (10 μM) and measured cAMP and renin release. Stimulation of the JG cell CaSR with the calcimimetic cinacalcet (1 μM) resulted in decreased cAMP from a basal of 1.13 ± 0.14 to 0.69 ± 0.08 pM/mg protein (P < 0.001) and in renin release from 0.89 ± 0.16 to 0.38 ± 0.08 μg ANG I/ml·h−1·mg protein−1 (P < 0.001). However, the addition of 8-MM-IBMX with cinacalcet returned both cAMP (1.10 ± 0.19 pM/mg protein) and renin (0.57 ± 0.16 μg ANG I/ml·h−1·mg protein−1) to basal levels. Similar results were obtained with vinpocetine, and also with W-7. Combining 8-MM-IBMX and W-7 had no additive effect. To determine which PDE1 isoform is involved, we performed Western blot analysis for PDE1A, B, and C. Only Western blot analysis for PDE1C showed a characteristic band apparent at 80 kDa. Immunofluorescence showed cytoplasmic distribution of PDE1C and renin in the JG cells. In conclusion, PDE1C is expressed in isolated JG cells, and contributes to calcium's inhibitory modulation of renin release from JG cells. PMID:19741056

Turning sunlight into stone: the oxalate-carbonate pathway in a tropical tree ecosystem

NASA Astrophysics Data System (ADS)

Cailleau, G.; Braissant, O.; Verrecchia, E. P.

2011-07-01

An African oxalogenic tree, the iroko tree (Milicia excelsa), has the property to enhance carbonate precipitation in tropical oxisols, where such accumulations are not expected due to the acidic conditions in these types of soils. This uncommon process is linked to the oxalate-carbonate pathway, which increases soil pH through oxalate oxidation. In order to investigate the oxalate-carbonate pathway in the iroko system, fluxes of matter have been identified, described, and evaluated from field to microscopic scales. In the first centimeters of the soil profile, decaying of the organic matter allows the release of whewellite crystals, mainly due to the action of termites and saprophytic fungi. In addition, a concomitant flux of carbonate formed in wood tissues contributes to the carbonate flux and is identified as a direct consequence of wood feeding by termites. Nevertheless, calcite biomineralization of the tree is not a consequence of in situ oxalate consumption, but rather related to the oxalate oxidation inside the upper part of the soil. The consequence of this oxidation is the presence of carbonate ions in the soil solution pumped through the roots, leading to preferential mineralization of the roots and the trunk base. An ideal scenario for the iroko biomineralization and soil carbonate accumulation starts with oxalatization: as the iroko tree grows, the organic matter flux to the soil constitutes the litter, and an oxalate pool is formed on the forest ground. Then, wood rotting agents (mainly termites, saprophytic fungi, and bacteria) release significant amounts of oxalate crystals from decaying plant tissues. In addition, some of these agents are themselves producers of oxalate (e.g. fungi). Both processes contribute to a soil pool of "available" oxalate crystals. Oxalate consumption by oxalotrophic bacteria can then start. Carbonate and calcium ions present in the soil solution represent the end products of the oxalate-carbonate pathway. The solution is pumped through the roots, leading to carbonate precipitation. The main pools of carbon are clearly identified as the organic matter (the tree and its organic products), the oxalate crystals, and the various carbonate features. A functional model based on field observations and diagenetic investigations with δ13C signatures of the various compartments involved in the local carbon cycle is proposed. It suggests that the iroko ecosystem can act as a long-term carbon sink, as long as the calcium source is related to non-carbonate rocks. Consequently, this carbon sink, driven by the oxalate carbonate pathway around an iroko tree, constitutes a true carbon trapping ecosystem as defined by ecological theory.

Turning sunlight into stone: the oxalate-carbonate pathway in a tropical tree ecosystem

NASA Astrophysics Data System (ADS)

Cailleau, G.; Braissant, O.; Verrecchia, E. P.

2011-02-01

An African oxalogenic tree, the iroko tree (Milicia excelsa), has the property to enhance carbonate precipitation in tropical oxisols, where such accumulations are not expected due to the theoretical acidic conditions of these soils. This uncommon process is linked to the oxalate-carbonate pathway, which increases soil pH through oxalate oxidation. In order to investigate the oxalate-carbonate pathway in the iroko system, fluxes of matter have been identified, described, and evaluated from field to microscopic scales. In the first centimeters of the soil profile, decaying of the organic matter allows the release of whewellite crystals, mainly due to the action of termites and saprophytic fungi. Regarding the carbonate flux, another direct consequence of wood feeding is a concomitant flux of carbonate formed in wood tissues, which is not consumed by termites. Nevertheless, calcite biomineralization of the tree is not a consequence of in situ oxalate consumption, but rather related to the oxalate oxidation inside the upper part of the soil. The consequence of this oxidation is the presence of carbonate ions in the soil solution pumped through the roots, leading to preferential mineralization of the roots and the trunk base. An ideal scenario for the iroko biomineralization and soil carbonate accumulation starts with oxalatization: as the iroko tree grows, the organic matter flux to the soil constitutes the litter. Therefore, an oxalate pool is formed on the forest ground. Then, wood rotting gents (mainly termites, fungi, and bacteria) release significant amounts of oxalate crystals from decaying plant tissues. In addition some of these gents are themselves producers of oxalate (fungi). Both processes contribute to a soil pool of "available" oxalate crystals. Oxalate consumption by oxalotrophic bacteria can start. Carbonate and calcium ions present in the soil solution represent the end products of the oxalate-carbonate pathway. The solution is pumped through the roots, leading to carbonate precipitation. The main pools of carbon are clearly identified as the organic matter (the tree and its organic products), the oxalate crystals, and the various carbonate features. A functional model based on field observations and diagenetic investigations with δ13C signatures of the various compartments involved in the local carbon cycle is proposed. It suggests that the iroko ecosystem can act as a long-term carbon sink, as long as the calcium source is related to non-carbonate rocks. Consequently, this carbon sink, driven by the oxalate carbonate pathway around an iroko tree, constitutes a true carbon trapping ecosystem as define by the ecological theory.

Physiochemical properties of experimental nano-hybrid MTA

PubMed Central

Akhavan Zanjani, V; Tabari, K; Sheikh-Al-Eslamian, SM; Abrandabadi, AN

2017-01-01

Introduction: Development of new pulp capping agents has paved the way towards the preservation of pulp vitality, which is an important goal in restorative dentistry. This study sought to assess the calcium ion release, pH and setting of mineral trioxide aggregate (MTA) Angelus, an experimental formulation of nano-hybrid MTA containing nano-SiO2, nano-Al2O3 and nano-TiO2 and MTA Angelus plus nano-oxides. Methods: In this experimental study, five specimens from each material were placed in polypropylene tubes and immersed in a flask containing deionized distilled water. The quantity of calcium ions released into the solution from each material was measured at 15 minutes, one hour, and 24 hours by using atomic absorption spectroscopy. The pH of the solutions was measured by using a pH meter at the respective time points. The setting time was also assessed by using a Gilmore needle. Data were analyzed by using repeated measure ANOVA. Results: The quantity of released calcium ions was not significantly different among the groups (P=0.060). All materials were alkaline and the pH at 24 hours was significantly higher than the other two time points in all groups (P<0.001). The experimental group had the shortest and the MTA Angelus had the longest setting time. All materials were alkaline and capable of releasing calcium. Addition of nanoparticles to MTA Angelus significantly decreased the setting time but had no effect on the release of calcium ions or pH. Abbreviations: MTA = mineral trioxide aggregate, VPT = vital pulp therapy PMID:29075348

Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants.

PubMed

Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

2017-01-16

To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants. Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-β1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (r s : 0.76), platelet counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (r s : 0.78), leukocyte counts in PRP with TGF-β1 concentrations in PRG supernatants (r s : 0.76), and PDGF-BB concentrations with activating substances (r s : 0.72). Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.

Modulation of bicarbonate secretion in rabbit duodenum: the role of calcium.

PubMed

Hogan, D L; Yao, B; Isenberg, J I

1998-01-01

Surface epithelial bicarbonate secretion protects the proximal duodenum from acid peptic injury. Cyclic adenosine monophosphate and calcium serve as intracellular mediators of intestinal transport. Experiments were performed to examine whether calcium participates in duodenal bicarbonate transport. Stripped duodenal mucosa from rabbits was studied in Ussing chambers. HCO3- transport was stimulated by the calcium ionophore A23187, carbachol, vasoactive intestinal peptide, prostaglandin E2, dibutyryl-cyclic adenosine monophosphate, and electrical field stimulation. A23187 stimulated HCO3- secretion and Isc; tetrodotoxin failed to inhibit this effect. The calcium-channel blocker verapamil abolished HCO3- secretion stimulated by carbachol, vasoactive intestinal peptide, and electrical field stimulation, but failed to alter basal, prostaglandin E2- or dibutyryl-cyclic adenosine monophosphate-stimulated HCO3- secretion. Therefore, calcium is likely required during stimulation of duodenal epithelial HCO3- transport by carbachol, vasoactive intestinal peptide, and electrical field stimulation. Prostaglandin E2 and dibutyryl-cyclic adenosine monophosphate appear to activate duodenal HCO3- secretion by a calcium-independent pathway(s).

The physical properties and ion release of CPP-ACP-modified calcium silicate-based cements.

PubMed

Dawood, A E; Manton, D J; Parashos, P; Wong, Rhk; Palamara, Jea; Stanton, D P; Reynolds, E C

2015-12-01

This study investigated the physical properties and ion release of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)-modified calcium silicate-based cements (CSCs) and compared the properties of a trial mineral trioxide aggregate (MTA) with two commercially available CSCs, Biodentine(™) and Angelus(®) MTA. The setting time, solubility, compressive strength and Vickers surface microhardness of the three CSCs incorporated with 0%, 0.5%, 1.0%, 2.0% and 3.0% (w/w) CPP-ACP were investigated. Release of calcium (Ca(2+) ), phosphate ions (Pi ) and pH of the test cements were measured after 24, 72, 168 and 336 h of storage. The addition of up to 1.0% CPP-ACP into Biodentine(™) and 0.5% into the other cements did not adversely affect their physical properties except for the setting time. The addition of 0.5% CPP-ACP increased Ca(2+) released from Biodentine(™) (after 168 and 336 h), Angelus(®) MTA (after 168 h) and the trial MTA (after 72 h). The addition of 1.0-3.0% CPP-ACP increased Ca(2+) and Pi released from all the cements. Biodentine(™) released more Ca(2+) particularly in the early stages and showed shorter setting time and higher mechanical properties than the other cements. The mechanical properties of Angelus(®) MTA and the trial MTA were similar. All the cements produced highly alkaline storage solutions. Up to 1.0% CPP-ACP in Biodentine(™) improves Ca(2+) and Pi release and 0.5% CPP-ACP in Angelus(®) MTA and the trial MTA improves Ca(2+) release without altering the mechanical properties and solubility. The addition of CPP-ACP into CSCs prolonged the setting time. © 2015 Australian Dental Association.

Effect of soft drinks on the release of calcium from enamel surfaces.

PubMed

Rirattanapong, Praphasri; Vongsavan, Kadkao; Surarit, Rudee

2013-09-01

Continuous consumption of soft drinks is the main cause of potential oral health problems, including dental caries and erosion. The purpose of this study was to compare the effect of three different types of soft drinks on the release of calcium from the enamel surface of teeth. Forty bovine teeth were selected for the experiment. They were divided into four groups (n=10/group): Group 1 (Coke), Group 2 (Pepsi), Group 3 (Sprite), and Group 4 (distilled water, the control). The pH of each beverage was measured using a pH meter. The release of calcium ions was measured using an atomic absorption spectrophotometer at baseline, 15, 30, and 60 minutes. The results were assessed by analysis of variance and then by the Tukey test (p< 0.05). Coke, with a pH of 2.39, was the most acidic among the soft drinks. Coke, Pepsi, and Sprite showed no significant mean differences in the calcium released, but there was a significant mean difference of these soft drinks with distilled water at 60 minutes. We concluded that prolonged exposure to soft drinks could lead to significant enamel loss.

Reverse Micelle Mediated synthesis of Calcium Phosphate Nanocarriers for Controlled Release of Bovine Serum Albumin (BSA)

PubMed Central

Dasgupta, Sudip; Bandyopadhyay, Amit; Bose, Susmita

2010-01-01

Calcium phosphate (CaP) nanoparticle with calcium to phosphorus (Ca:P) molar ratio of 1.5:1 were synthesized using reverse micro emulsion. Ca(NO3)2.4H2O and H3PO4 were used as aqueous phase, cyclohexane as organic phase, and poly(oxyethylene)12 nonylphenol ether (NP-12) as surfactant. Depending on calcination temperature between 600 and 800 °C, CaP nanoparticle showed different phases calcium deficient hydroxyapatite (CDHA) and β-tricalcium phosphate (β-TCP), particle size between 48 and 69 nm, the BET specific average surface area between 73 m2/g and 57 m2/g. Bovine serum albumin (BSA) was used as a model protein to study loading and release behavior. Adsorptive property of BSA was investigated with the change in BET surface area of these nanoparticle and the pH of the suspension. At pH 7.5, maximum amount of BSA was adsorbed onto CaP nanoparticle. The release kinetics of BSA showed a gradual time dependent increase at pH 4.0 and 6.0 buffer solutions. However, the amount of released protein was significantly smaller at pH 7.2. BSA release rate also varied depending on the presence of different phases of CaPs in the system, β-TCP or CDHA. These results suggest that BSA protein release rate can be controlled by changing particle size, surface area and phase composition of CaP nanocarriers. PMID:19435617

Signal mass and Ca²⁺ kinetics in local calcium events: a modeling study.

PubMed

Baran, Irina; Ganea, Constanta; Ungureanu, Raluca; Tofolean, Ioana Teodora

2012-02-01

We use a detailed modeling formalism based on numerical simulations of local calcium release events where the blurring of the image, the presence of diffusional barriers provided by large organelles situated close to the release site, as well as the variable position of the scan line with respect to the release site are taken into consideration. We have investigated the effect of the fluorescence noise fluctuations on the accuracy in computing the signal mass from linescan recordings and obtained a quantitative description of both the signal mass and the local increase in the free Ca(2+) level as a function of the release current, the release duration and the orientation of the scan line, for three different levels of noise magnitudes. The model could provide a very good fit to a wide set of available experimental data regarding the signal mass of puffs visualized by fluorescence microscopy in the Xenopus oocyte loaded with 40 μM Oregon Green-1 in the absence of the calcium chelator EGTA. Numerical simulations also predict the amplitude and the kinetics of calcium signals evolving in the absence of the indicator, and indicate that sub-maximal activation of IP(3) receptors could produce in average levels of about 2 μM and 0.4 μM free Ca(2+) close to a release site located in the animal or in the vegetal hemisphere, respectively, whereas the maximal levels reached in more rare events could be 11 μM and 4 μM, respectively.

Stimulation by atropine of acetylcholine release and synthesis in cortical slices from rat brain

PubMed Central

Molenaar, P. C.; Polak, R. L.

1970-01-01

1. Cortical slices from rat brain were incubated in media containing the irreversible cholinesterase inhibitor soman and a high KCl concentration, and the release and synthesis of acetylcholine (ACh) were determined. 2. Atropine enhanced the release and synthesis of ACh. 3. Tetrodotoxin, a substance which blocks nervous conduction, did not influence the release and synthesis of ACh, in the absence or in the presence of atropine. Therefore the nerve endings are probably the site at which atropine acts when stimulating the release and synthesis of ACh. 4. Pretreatment of the slices with botulinum type A toxin partially blocked the release and synthesis of ACh and reduced the extra amounts of ACh released and synthesized under the influence of atropine. 5. Lowering the calcium or raising the magnesium concentration in the incubation medium reduced the release and synthesis of ACh and their enhancement by atropine. 6. Physostigmine decreased the total extractable ACh content of the slices during incubation in a 25 mM KCl containing medium. This decrease was nearly prevented when the release and synthesis of ACh were inhibited by omission of the calcium ions from the medium, but was enhanced by atropine. 7. The observations made with pretreatment by botulinum type A toxin, with changes in the calcium and magnesium concentration as well as with physostigmine, all support the theory that it is primarily the release of ACh which is enhanced by atropine and that its stimulating action on the synthesis results from the increased release. PMID:5497792

Development of modified in situ gelling oral liquid sustained release formulation of dextromethorphan.

PubMed

El Maghraby, Gamal M; Elzayat, Ehab M; Alanazi, Fars K

2012-08-01

Alternative strategies are being employed to develop liquid oral sustained release formulation. These included ion exchange resin, sustained release suspensions and in situ gelling systems. The later mainly utilizes alginate solutions that form gels upon contact with calcium which may be administered separately or included in the alginate solution as citrate complex. This complex liberates calcium in the stomach with subsequent gellation. The formed gel can break after gastric emptying leading to dose dumping. Development of modified in situ gelling system which sustain dextromethorphan release in the stomach and intestine. Solutions containing alginate with calcium chloride and sodium citrate were initially prepared to select the formulation sustaining the release in the stomach. The best formulation was combined with chitosan. All formulations were characterized with respect to flow, gelling capacity, gelling strength and drug release. Increasing the concentration of alginate increased the gelling capacity and strength and reduced the rate of drug release in gastric conditions with 2% w/v alginate being the best formulation. However, these formulations failed to sustain the release in the intestinal conditions. Incorporation of chitosan with alginate increased the gelling capacity and strength and reduced the rate of drug release compared to alginate only system. The effect was optimum in formulation containing 1.5% w/v chitosan. The sustained release pattern was maintained both in the gastric and intestinal conditions and was comparable to that obtained from the marketed product. Alginate-chitosan based in situ gelling system is promising for developing liquid oral sustained release.

Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

PubMed Central

López Soto, Eduardo Javier; Agosti, Francina; Cabral, Agustina; Mustafa, Emilio Roman; Damonte, Valentina Martínez; Gandini, Maria Alejandra; Rodríguez, Silvia; Castrogiovanni, Daniel; Felix, Ricardo; Perelló, Mario

2015-01-01

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G protein–coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the voltage-gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and agonist-dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons. PMID:26283199

ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

PubMed Central

Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

2011-01-01

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

Incorporation of casein phosphopeptide-amorphous calcium phosphate into a glass-ionomer cement.

PubMed

Mazzaoui, S A; Burrow, M F; Tyas, M J; Dashper, S G; Eakins, D; Reynolds, E C

2003-11-01

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes have been shown to prevent demineralization and promote remineralization of enamel subsurface lesions in animal and in situ caries models. The aim of this study was to determine the effect of incorporating CPP-ACP into a self-cured glass-ionomer cement (GIC). Incorporation of 1.56% w/w CPP-ACP into the GIC significantly increased microtensile bond strength (33%) and compressive strength (23%) and significantly enhanced the release of calcium, phosphate, and fluoride ions at neutral and acidic pH. MALDI mass spectrometry also showed casein phosphopeptides from the CPP-ACP nanocomplexes to be released. The release of CPP-ACP and fluoride from the CPP-ACP-containing GIC was associated with enhanced protection of the adjacent dentin during acid challenge in vitro.

Treatment of mcf-7 breast cancer cells with a red grape wine polyphenol fraction results in disruption of calcium homeostasis and cell cycle arrest causing selective cytotoxicity.

PubMed

Hakimuddin, Fatima; Paliyath, Gopinadhan; Meckling, Kelly

2006-10-04

Food components influence the physiology by modulating gene expression and biochemical pathways within the human body. The disease-preventive roles of several fruit and vegetable components have been related to such properties. Polyphenolic components such as flavonoids are strong antioxidants and induce the expression of several xenobiotic-detoxifying enzymes. The mechanism of selective cytotoxicity induced by red grape wine polyphenols against MCF-7 breast cancer cells was investigated in relation to their interference with calcium homeostasis. MCF-7 cells showed an increase in cytosolic calcium levels within 10 min of treatment with the polyphenols. Immunohistochemical localization of calmodulin with secondary gold-labeled antibodies showed similar levels of gold labeling in both MCF-7 cells and the spontaneously immortalized, normal MCF-10A cell line. MCF-7 cells treated with the red wine polyphenol fraction (RWPF) showed swelling of endoplasmic reticulum, dissolution of the nucleus, and loss of plasma membrane integrity as well as reduced mitochondrial membrane potential. These cells were arrested at the G2/M interphase. By contrast, MCF-10A cells did not show such changes after RWPF treatment. The results suggest that polyphenol-induced calcium release may disrupt mitochondrial function and cause membrane damage, resulting in selective cytotoxicity toward MCF-7 cells. This property could further be developed toward breast cancer prevention strategies either independently or in conjunction with conventional prevention therapies where a positive drug-nutrient interaction can be demonstrated.

Effect of Reaction Pathway on the Extent and Mechanism of Uranium(VI) Immobilization with Calcium and Phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mehta, Vrajesh S.; Maillot, Fabien; Wang, Zheming

Phosphate addition to subsurface environments contaminated with uranium can be used as an in situ remediation approach. Batch experiments were conducted to evaluate the dependence of the extent and mechanism of uranium uptake on the pathway for reaction with calcium phosphates. At pH 4.0 and 6.0 uranium uptake occurred via autunite (Ca(UO2)(PO4)3) precipitation irrespective of the starting forms of calcium and phosphate. At pH 7.5, the uptake mechanism depended on the nature of the calcium and phosphate. When dissolved uranium, calcium, and phosphate were added simultaneously, uranium was structurally incorporated into a newly formed amorphous calcium phosphate solid. Adsorption wasmore » the dominant removal mechanism for uranium contacted with pre-formed amorphous calcium phosphate solids,. When U(VI) was added to a suspension containing amorphous calcium phosphate solids as well as dissolved calcium and phosphate, then removal occurred through precipitation (57±4 %) of autunite and adsorption (43±4 %) onto calcium phosphate. The solid phase speciation of the uranium was determined using X-ray absorption spectroscopy and laser induced fluorescence spectroscopy. Dissolved uranium, calcium, and phosphate concentrations with saturation index calculations helped identify removal mechanisms and determine thermodynamically favorable solid phases.« less

Cardiac cell: a biological laser?

PubMed

Chorvat, D; Chorvatova, A

2008-04-01

We present a new concept of cardiac cells based on an analogy with lasers, practical implementations of quantum resonators. In this concept, each cardiac cell comprises a network of independent nodes, characterised by a set of discrete energy levels and certain transition probabilities between them. Interaction between the nodes is given by threshold-limited energy transfer, leading to quantum-like behaviour of the whole network. We propose that in cardiomyocytes, during each excitation-contraction coupling cycle, stochastic calcium release and the unitary properties of ionic channels constitute an analogue to laser active medium prone to "population inversion" and "spontaneous emission" phenomena. This medium, when powered by an incoming threshold-reaching voltage discharge in the form of an action potential, responds to the calcium influx through L-type calcium channels by stimulated emission of Ca2+ ions in a coherent, synchronised and amplified release process known as calcium-induced calcium release. In parallel, phosphorylation-stimulated molecular amplification in protein cascades adds tuneable features to the cells. In this framework, the heart can be viewed as a coherent network of synchronously firing cardiomyocytes behaving as pulsed laser-like amplifiers, coupled to pulse-generating pacemaker master-oscillators. The concept brings a new viewpoint on cardiac diseases as possible alterations of "cell lasing" properties.

Interaction of zinc with dental mineral.

PubMed

Ingram, G S; Horay, C P; Stead, W J

1992-01-01

As some currently available toothpastes contain zinc compounds, the reaction of zinc with dental mineral and its effect on crystal growth rates were studied using three synthetic calcium-deficient hydroxyapatites (HAP) as being representative of dental mineral. Zinc was readily acquired by all HAP samples in the absence of added calcium, the amount adsorbed being proportional to the HAP surface area; about 9 mumol Zn/m2 was adsorbed at high zinc concentrations. As zinc was acquired, calcium was released, consistent with 1:1 Ca:Zn exchange. Soluble calcium reduced zinc uptake and similarly, calcium post-treatment released zinc. Pretreatment of HAP with 0.5 mM zinc reduced its subsequent ability to undergo seeded crystal growth, as did extracts of a toothpaste containing 0.5% zinc citrate, even in the presence of saliva. The reverse reaction, i.e. displacement of adsorbed zinc by salivary levels of calcium, however, indicates the mechanism by which zinc can reduce calculus formation in vivo by inhibiting plaque mineralisation without adversely affecting the anti-caries effects of fluoride.

Effects of supplemental calcium and vitamin D on the APC/β-catenin pathway in the normal colorectal mucosa of colorectal adenoma patients.

PubMed

Liu, Siyu; Barry, Elizabeth L; Baron, John A; Rutherford, Robin E; Seabrook, March E; Bostick, Roberd M

2017-02-01

APC/β-catenin pathway malfunction is a common and early event in colorectal carcinogenesis. To assess calcium and vitamin D effects on the APC/β-catenin pathway in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, nested within a larger randomized, double-blind, placebo-controlled, partial 2 × 2 factorial chemoprevention clinical trial of supplemental calcium (1200 mg daily) and vitamin D (1000 IU daily), alone and in combination versus placebo, we assessed APC, β-catenin, and E-cadherin expression in colon crypts in normal-appearing rectal mucosa biopsies from 104 participants at baseline and 1-yr follow up using standardized, automated immunohistochemistry and quantitative image analysis. For vitamin D versus no vitamin D, the ratio of APC expression to β-catenin expression in the upper 40% (differentiation zone) of crypts (APC/β-catenin score) increased by 28% (P = 0.02), for calcium versus no calcium it increased by 1% (P = 0.88), and for vitamin D + calcium versus calcium by 35% (P = 0.01). Total E-cadherin expression increased by 7% (P = 0.35) for vitamin D versus no vitamin D, 8% (P = 0.31) for calcium versus no calcium, and 12% (P = 0.21) for vitamin D + calcium versus calcium. These results support (i) that vitamin D, alone or in combination with calcium, may modify APC, β-catenin, and E-cadherin expression in humans in directions hypothesized to reduce risk for colorectal neoplasms; (ii) vitamin D as a potential chemopreventive agent against colorectal neoplasms; and (iii) the potential of APC, β-catenin, and E-cadherin expression as treatable, pre-neoplastic risk biomarkers for colorectal neoplasms. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Wax-incorporated emulsion gel beads of calcium pectinate for intragastric floating drug delivery.

PubMed

Sriamornsak, Pornsak; Asavapichayont, Panida; Nunthanid, Jurairat; Luangtana-Anan, Manee; Limmatvapirat, Sontaya; Piriyaprasarth, Suchada

2008-01-01

The purpose of this study was to prepare wax-incorporated pectin-based emulsion gel beads using a modified emulsion-gelation method. The waxes in pectin-olive oil mixtures containing a model drug, metronidazole, were hot-melted, homogenized and then extruded into calcium chloride solution. The beads formed were separated, washed with distilled water and dried for 12 h. The influence of various types and amounts of wax on floating and drug release behavior of emulsion gel beads of calcium pectinate was investigated. The drug-loaded gel beads were found to float on simulated gastric fluid if the sufficient amount of oil was used. Incorporation of wax into the emulsion gel beads affected the drug release. Water-soluble wax (i.e. polyethylene glycol) increased the drug release while other water-insoluble waxes (i.e. glyceryl monostearate, stearyl alcohol, carnauba wax, spermaceti wax and white wax) significantly retarded the drug release. Different waxes had a slight effect on the drug release. However, the increased amount of incorporated wax in the formulations significantly sustained the drug release while the beads remained floating. The results suggest that wax-incorporated emulsion gel beads could be used as a carrier for intragastric floating drug delivery.

Localized intracellular calcium signaling in muscle: calcium sparks and calcium quarks.

PubMed

Niggli, E

1999-01-01

Subcellularly localized Ca2+ signals in cardiac and skeletal muscle have recently been identified as elementary Ca2+ signaling events. The signals, termed Ca2+ sparks and Ca2+ quarks, represent openings of Ca2+ release channels located in the membrane of the sarcoplasmic reticulum (SR). In cardiac muscle, the revolutionary discovery of Ca2+ sparks has allowed the development of a fundamentally different concept for the amplification of Ca2+ signals by Ca(2+)-induced Ca2+ release. In such a system, a graded amplification of the triggering Ca2+ signal entering the myocyte via L-type Ca2+ channels is accomplished by a recruitment process whereby individual SR Ca2+ release units are locally controlled by L-type Ca2+ channels. In skeletal muscle, the initial SR Ca2+ release is governed by voltage-sensors but subsequently activates additional Ca2+ sparks by Ca(2+)-induced Ca2+ release from the SR. Results from studies on elementary Ca2+ release events will improve our knowledge of muscle Ca2+ signaling at all levels of complexity, from the molecule to normal cellular function, and from the regulation of cardiac and skeletal muscle force to the pathophysiology of excitation-contraction coupling.

Calcium intake: good for the bones but bad for the heart? An analysis of clinical studies.

PubMed

Lima, Guilherme Alcantara Cunha; Lima, Priscilla Damião Araújo; Barros, Maria da Glória Costa Reis Monteiro de; Vardiero, Lívia Paiva; Melo, Elisa Fernandes de; Paranhos-Neto, Francisco de Paula; Madeira, Miguel; Farias, Maria Lucia Fleiuss de

2016-06-01

The proper dietary calcium intake and calcium supplementation, when indicated, are important factors in the acquisition of peak bone mass during youth and in the prevention of fractures in old age. In addition to its deposition in bone, calcium confers an increase in its resistance and exhibits important activities in different enzymatic pathways in the body (e.g., neural, hormonal, muscle-related and blood clotting pathways). Thus, calcium supplementation can directly or indirectly affect important functions in the body, such as the control of blood pressure, plasma glucose, body weight, lipid profile and endothelial function. Since one publication reported increased cardiovascular risk due to calcium supplementation, many researchers have studied whether this risk actually exists; the results are conflicting, and the involved mechanisms are uncertain. However, studies that have evaluated the influence of the consumption of foods rich in calcium have reported no increase in the cardiovascular risk, which suggests that nutritional intake should be prioritized as a method for supplementation and that the use of calcium supplements should be reserved for patients who truly need supplementation and are unable to achieve the recommended daily nutritional intake of calcium.

Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess1[C][W][OA

PubMed Central

González, Alberto; Cabrera, M. de los Ángeles; Henríquez, M. Josefa; Contreras, Rodrigo A.; Morales, Bernardo; Moenne, Alejandra

2012-01-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H2O2) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H2O2, ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H2O2 increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H2O2 accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H2O2. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H2O2, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases. PMID:22234999

Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium-dependent protein kinases in Ulva compressa exposed to copper excess.

PubMed

González, Alberto; Cabrera, M de Los Ángeles; Henríquez, M Josefa; Contreras, Rodrigo A; Morales, Bernardo; Moenne, Alejandra

2012-03-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H(2)O(2), ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H(2)O(2) increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H(2)O(2) accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H(2)O(2). In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H(2)O(2), and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.

Calcium Activates Nedd4 E3 Ubiquitin Ligases by Releasing the C2 Domain-mediated Auto-inhibition*

PubMed Central

Wang, Jian; Peng, Qisheng; Lin, Qiong; Childress, Chandra; Carey, David; Yang, Wannian

2010-01-01

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-β. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination. PMID:20172859

Calcium/calmodulin and cAMP/protein kinase-A pathways regulate sperm motility in the stallion.

PubMed

Lasko, Jodi; Schlingmann, Karen; Klocke, Ann; Mengel, Grace Ann; Turner, Regina

2012-06-01

In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II β were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acrosome and flagellar principal piece. Additionally, inhibition of either calmodulin or protein kinase-A significantly reduced sperm motility without affecting viability. Following inhibition of calmodulin, motility was not restored with agonists of the cyclic AMP/protein kinase-A pathway. These data suggest that calcium/calmodulin and cyclic AMP/protein kinase-A pathways are involved in the regulation of stallion sperm motility. The failure of cyclic AMP/protein kinase-A agonists to restore motility of calmodulin inhibited sperm suggests that both pathways may be required to support normal motility. Copyright © 2012 Elsevier B.V. All rights reserved.

Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells.

PubMed

Elgueta, Claudio; Leroy, Felix; Vielma, Alex H; Schmachtenberg, Oliver; Palacios, Adrian G

2018-02-15

A17 amacrine cells are an important part of the scotopic pathway. Their synaptic varicosities receive glutamatergic inputs from rod bipolar cells (RBC) and release GABA onto the same RBC terminal, forming a reciprocal feedback that shapes RBC depolarization. Here, using patch-clamp recordings, we characterized electrical coupling between A17 cells of the rat retina and report the presence of strongly interconnected and non-coupled A17 cells. In coupled A17 cells, evoked currents preferentially flow out of the cell through GJs and cross-synchronization of presynaptic signals in a pair of A17 cells is correlated to their coupling degree. Moreover, we demonstrate that stimulation of one A17 cell can induce electrical and calcium transients in neighboring A17 cells, thus confirming a functional flow of information through electrical synapses in the A17 coupled network. Finally, blocking GJs caused a strong decrease in the amplitude of the inhibitory feedback onto RBCs. We therefore propose that electrical coupling between A17 cells enhances feedback onto RBCs by synchronizing and facilitating GABA release from inhibitory varicosities surrounding each RBC axon terminal. GJs between A17 cells are therefore critical in shaping the visual flow through the scotopic pathway.

G protein modulation of CaV2 voltage-gated calcium channels.

PubMed

Currie, Kevin P M

2010-01-01

Voltage-gated Ca(2+) channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca(2+) . As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of CaV2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α1 subunit of CaV2 channels. This article will review the basics of Ca(2+) -channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.

The quantal nature of calcium release to caffeine in single smooth muscle cells results from activation of the sarcoplasmic reticulum Ca(2+)-ATPase.

PubMed

Steenbergen, J M; Fay, F S

1996-01-26

Calcium release from intracellular stores occurs in a graded manner in response to increasing concentrations of either inositol 1,4,5-trisphosphate or caffeine. To investigate the mechanism responsible for this quantal release phenomenon, [Ca2+] changes inside intracellular stores in isolated single smooth muscle cells were monitored with mag-fura 2. Following permeabilization with saponin or alpha-toxin the dye, loaded via its acetoxymethyl ester, was predominantly trapped in the sarcoplasmic reticulum (SR). Low caffeine concentrations in the absence of ATP induced only partial Ca2+ release; however, after inhibiting the calcium pump with thapsigargin the same stimulus released twice as much Ca2+. When the SR Ca(2+)-ATPase was rendered non-functional by depleting its "ATP pool," submaximal caffeine doses almost fully emptied the stores of Ca2+. We conclude that quantal release of Ca2+ in response to caffeine in these smooth muscle cells is largely due to the activity of the SR Ca(2+)-ATPase, which appears to return a portion of the released Ca2+ back to the SR, even in the absence of ATP. Apparently the SR Ca(2+)-ATPase is fueled by ATP, which is either compartmentalized or bound to the SR.

Systems Biology Approach Reveals a Calcium-Dependent Mechanism for Basal Toxicity in Daphnia magna.

PubMed

Antczak, Philipp; White, Thomas A; Giri, Anirudha; Michelangeli, Francesco; Viant, Mark R; Cronin, Mark T D; Vulpe, Chris; Falciani, Francesco

2015-09-15

The expanding diversity and ever increasing amounts of man-made chemicals discharged to the environment pose largely unknown hazards to ecosystem and human health. The concept of adverse outcome pathways (AOPs) emerged as a comprehensive framework for risk assessment. However, the limited mechanistic information available for most chemicals and a lack of biological pathway annotation in many species represent significant challenges to effective implementation of this approach. Here, a systems level, multistep modeling strategy demonstrates how to integrate information on chemical structure with mechanistic insight from genomic studies, and phenotypic effects to define a putative adverse outcome pathway. Results indicated that transcriptional changes indicative of intracellular calcium mobilization were significantly overrepresented in Daphnia magna (DM) exposed to sublethal doses of presumed narcotic chemicals with log Kow ≥ 1.8. Treatment of DM with a calcium ATPase pump inhibitor substantially recapitulated the common transcriptional changes. We hypothesize that calcium mobilization is a potential key molecular initiating event in DM basal (narcosis) toxicity. Heart beat rate analysis and metabolome analysis indicated sublethal effects consistent with perturbations of calcium preceding overt acute toxicity. Together, the results indicate that altered calcium homeostasis may be a key early event in basal toxicity or narcosis induced by lipophilic compounds.

Releasing-addition method for the flame-photometric determination of calcium in thermal waters

USGS Publications Warehouse

Rowe, J.J.

1963-01-01

Study of the interferences of silica and sulfate in the flame-photometric determination of calcium in thermal waters has led to the development of a method requiring no prior chemical separations. The interference effects of silica, sulfate, potassium, sodium, aluminum, and phosphate are overcome by an addition technique coupled with the use of magnesium as a releasing agent. ?? 1963.

Burst discharges in neurons of the thalamic reticular nucleus are shaped by calcium-induced calcium release.

PubMed

Coulon, Philippe; Herr, David; Kanyshkova, Tatyana; Meuth, Patrick; Budde, Thomas; Pape, Hans-Christian

2009-01-01

The nucleus reticularis thalami (NRT) is a layer of inhibitory neurons that surrounds the dorsal thalamus. It appears to be the 'pacemaker' of certain forms of slow oscillations in the thalamus and was proposed to be a key determinant of the internal attentional searchlight as well as the origin of hypersynchronous activity during absence seizures. Neurons of the NRT exhibit a transient depolarization termed low threshold spike (LTS) following sustained hyperpolarization. This is caused by the activation of low-voltage-activated Ca2+ channels (LVACC). Although the role of these channels in thalamocortical oscillations was studied in great detail, little is known about the downstream intracellular Ca2+ signalling pathways and their feedback onto the oscillations. A signalling triad consisting of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), Ca2+ activated K+ channels (SK2), and LVACC is active in dendrites of NRT neurons and shapes rhythmic oscillations. The aim of our study was to find out (i) if and how Ca2+-induced Ca2+ release (CICR) via ryanodine receptors (RyR) can be evoked in NRT neurons and (ii) how the released Ca2+ affects burst activity. Combining electrophysiological, immunohistochemical, and two-photon Ca2+ imaging techniques, we show that CICR in NRT neurons takes place by a cell-type specific coupling of LVACC and RyR. CICR could be evoked by the application of caffeine, by activation of LVACC, or by repetitive LTS generation. During the latter, CICR contributed 30% to the resulting build-up of [Ca2+]i. CICR was abolished by cyclopiazonic acid, a specific blocker for SERCA, or by high concentrations of ryanodine (50 microM). Unlike other thalamic nuclei, in the NRT the activation of high-voltage-activated Ca2+ channels failed to evoke CICR. While action potentials contributed little to the build-up of [Ca2+]i upon repetitive LTS generation, the Ca2+ released via RyR significantly reduced the number of action potentials during an LTS and reduced the neurons' low threshold activity, thus potentially reducing hypersynchronicity. This effect persisted in the presence of the SK2 channel blocker apamin. We conclude that the activation of LVACC specifically causes CICR via RyR in neurons of the NRT, thereby adding a Ca2+-dependent intracellular route to the mechanisms determining rhythmic oscillatory bursting in this nucleus.

Leukotriene C4 production by murine mast cells: evidence of a role for extracellular leukotriene A4.

PubMed Central

Dahinden, C A; Clancy, R M; Gross, M; Chiller, J M; Hugli, T E

1985-01-01

The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell-bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes. PMID:2995976

Imperatoxin a enhances Ca(2+) release in developing skeletal muscle containing ryanodine receptor type 3.

PubMed Central

Nabhani, Thomas; Zhu, Xinsheng; Simeoni, Ilenia; Sorrentino, Vincenzo; Valdivia, Héctor H; García, Jesús

2002-01-01

Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [(3)H]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor. PMID:11867448

Calcium at fertilization and in early development

PubMed Central

Whitaker, Michael

2012-01-01

Fertilization calcium waves are introduced and the evidence from which we can infer general mechanisms of these waves is presented. The two main classes of hypothesis put forward to explain the generation of the fertilization calcium wave are set out and it is concluded that initiation of the fertilization calcium wave can be most generally explained in inverterbrates by a mechanism in which an activating substance enters the egg from the sperm on sperm-egg fusion, activating the egg by stimulating phospholipase C activation through a src family kinase pathway and in mammals by the diffusion of a sperm-specific phospholipase C from sperm to egg on sperm-egg fusion. The fertilization calcium wave is then set into the context of cell cycle control and the mechanism of repetitive calcium spiking in mammalian eggs is investigated. Evidence that calcium signals control cell division in early embryos is reviewed, and it is concluded that calcium signals are essential at all three stages of cell division in early embryos. Evidence that phosphoinositide signalling pathways control the resumption of meiosis during oocyte maturation is considered. It is concluded on balance that the evidence points to a need for phosphoinositide/calcium signalling during resumption of meiosis. Changes to the calcium signalling machinery occur during meiosis to enable the production of a calcium wave in the mature oocyte when it is fertilized; evidence that the shape and structure of the endoplasmic reticulum alters dynamically during maturation and after fertilization is reviewed and the link between ER dynamics and the cytoskeleton is discussed. There is evidence that calcium signalling plays a key part in the development of patterning in early embryos. Morphogenesis in ascidian, frog and zebrafish embryos is briefly described to provide the developmental context in which calcium signals act. Intracellular calcium waves that may play a role in axis formation in ascidian are discussed. Evidence that the Wingless/calcium signalling pathway is a strong ventralizing signal in Xenopus, mediated by phoshoinositide signalling is adumbrated. The central role that calcium channels play in morphogenetic movements during gastrulation and in ectodermal and mesodermal gene expression during late gastrulation is demonstrated. Experiments in zebrafish provide a strong indication that calcium signals are essential for pattern formation and organogenesis. PMID:16371595

Calcium-silicate mesoporous nanoparticles loaded with chlorhexidine for both anti- Enterococcus faecalis and mineralization properties.

PubMed

Fan, Wei; Li, Yanyun; Sun, Qing; Ma, Tengjiao; Fan, Bing

2016-10-21

In infected periapical tissues, Enterococcus faecalis is one of the most common dominant bacteria. Chlorhexidine has been proved to show strong antibacterial ability against E. faecalis but is ineffective in promoting mineralization for tissues around root apex. Mesoporous calcium-silicate nanoparticles are newly synthesized biomaterials with excellent ability to promote mineralization and carry-release bioactive molecules in a controlled manner. In this study, mesoporous calcium-silicate nanoparticles were functionalized with chlorhexidine and their releasing profile, antibacterial ability, effect on cell proliferation and in vitro mineralization property were evaluated. The chlorhexidine was successfully incorporated into mesoporous calcium-silicate nanoparticles by a mixing-coupling method. The new material could release chlorhexidine as well as Ca 2+ and SiO 3 2- in a sustained manner with an alkaline pH value under different conditions. The antimicrobial ability against planktonic E. faecalis was dramatically improved after chlorhexidine incorporation. The nanoparticles with chlorhexidine showed no negative effect on cell proliferation with low concentrations. On dentin slices, the new synthesized material demonstrated a similar inhibitory effect on E. faecalis as the chlorhexidine. After being immersed in SBF for 9 days, numerous apatite crystals could be observed on surfaces of the material tablets. Mesoporous calcium-silicate nanoparticles loaded with chlorhexidine exhibited release of ions and chlorhexidine, low cytotoxicity, excellent antibacterial ability and in vitro mineralization. This material could be developed into a new effective intra-canal medication in dentistry or a new bone defect filling material for infected bone defects.

Bayesian analysis of the kinetics of quantal transmitter secretion at the neuromuscular junction.

PubMed

Saveliev, Anatoly; Khuzakhmetova, Venera; Samigullin, Dmitry; Skorinkin, Andrey; Kovyazina, Irina; Nikolsky, Eugeny; Bukharaeva, Ellya

2015-10-01

The timing of transmitter release from nerve endings is considered nowadays as one of the factors determining the plasticity and efficacy of synaptic transmission. In the neuromuscular junction, the moments of release of individual acetylcholine quanta are related to the synaptic delays of uniquantal endplate currents recorded under conditions of lowered extracellular calcium. Using Bayesian modelling, we performed a statistical analysis of synaptic delays in mouse neuromuscular junction with different patterns of rhythmic nerve stimulation and when the entry of calcium ions into the nerve terminal was modified. We have obtained a statistical model of the release timing which is represented as the summation of two independent statistical distributions. The first of these is the exponentially modified Gaussian distribution. The mixture of normal and exponential components in this distribution can be interpreted as a two-stage mechanism of early and late periods of phasic synchronous secretion. The parameters of this distribution depend on both the stimulation frequency of the motor nerve and the calcium ions' entry conditions. The second distribution was modelled as quasi-uniform, with parameters independent of nerve stimulation frequency and calcium entry. Two different probability density functions for the distribution of synaptic delays suggest at least two independent processes controlling the time course of secretion, one of them potentially involving two stages. The relative contribution of these processes to the total number of mediator quanta released depends differently on the motor nerve stimulation pattern and on calcium ion entry into nerve endings.

Rapid frequency‐dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes

PubMed Central

Wüst, Rob C. I.; Helmes, Michiel; Martin, Jody L.; van der Wardt, Thomas J. T.; Musters, René J. P.; van der Velden, Jolanda

2017-01-01

Key points Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle.The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown.Rapid stimulation frequency‐dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency.These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. Abstract Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)‐based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1–4 Hz) and external calcium concentrations (1.8–3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura‐4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium–calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and, paradoxically, in an acceleration of Ca2+ release. In conclusion: rapid increases in [Ca2+]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat‐to‐beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering. PMID:28028811

A novel pH-responsive hydrogel-based on calcium alginate engineered by the previous formation of polyelectrolyte complexes (PECs) intended to vaginal administration.

PubMed

Ferreira, Natália Noronha; Perez, Taciane Alvarenga; Pedreiro, Liliane Neves; Prezotti, Fabíola Garavello; Boni, Fernanda Isadora; Cardoso, Valéria Maria de Oliveira; Venâncio, Tiago; Gremião, Maria Palmira Daflon

2017-10-01

This work aimed to develop a calcium alginate hydrogel as a pH responsive delivery system for polymyxin B (PMX) sustained-release through the vaginal route. Two samples of sodium alginate from different suppliers were characterized. The molecular weight and M/G ratio determined were, approximately, 107 KDa and 1.93 for alginate_S and 32 KDa and 1.36 for alginate_V. Polymer rheological investigations were further performed through the preparation of hydrogels. Alginate_V was selected for subsequent incorporation of PMX due to the acquisition of pseudoplastic viscous system able to acquiring a differential structure in simulated vaginal microenvironment (pH 4.5). The PMX-loaded hydrogel (hydrogel_PMX) was engineered based on polyelectrolyte complexes (PECs) formation between alginate and PMX followed by crosslinking with calcium chloride. This system exhibited a morphology with variable pore sizes, ranging from 100 to 200 μm and adequate syringeability. The hydrogel liquid uptake ability in an acid environment was minimized by the previous PECs formation. In vitro tests evidenced the hydrogels mucoadhesiveness. PMX release was pH-dependent and the system was able to sustain the release up to 6 days. A burst release was observed at pH 7.4 and drug release was driven by an anomalous transport, as determined by the Korsmeyer-Peppas model. At pH 4.5, drug release correlated with Weibull model and drug transport was driven by Fickian diffusion. The calcium alginate hydrogels engineered by the previous formation of PECs showed to be a promising platform for sustained release of cationic drugs through vaginal administration.

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

PubMed Central

Alvarez-Lacalle, Enric; Cantalapiedra, Inma R.; Peñaranda, Angelina; Cinca, Juan; Hove-Madsen, Leif; Echebarria, Blas

2013-01-01

Background Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive. Methodology/Principal Findings To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s. Conclusions/Significance We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present. PMID:23390511

Polyamine regulates tolerance to water stress in leaves of white clover associated with antioxidant defense and dehydrin genes via involvement in calcium messenger system and hydrogen peroxide signaling

PubMed Central

Li, Zhou; Zhang, Yan; Peng, Dandan; Wang, Xiaojuan; Peng, Yan; He, Xiaoshuang; Zhang, Xinquan; Ma, Xiao; Huang, Linkai; Yan, Yanhong

2015-01-01

Endogenous polyamine (PA) may play a critical role in tolerance to water stress in plants acting as a signaling molecule activator. Water stress caused increases in endogenous PA content in leaves, including putrescine (Put), spermidine (Spd), and spermine (Spm). Exogenous application of Spd could induce the instantaneous H2O2 burst and accumulation of cytosolic free Ca2+, and activate NADPH oxidase and CDPK gene expression in cells. To a great extent, PA biosynthetic inhibitor reduced the water stress-induced H2O2 accumulation, free cytosolic Ca2+ release, antioxidant enzyme activities and genes expression leading to aggravate water stress-induced oxidative damage, while these suppressing effects were alleviated by the addition of exogenous Spd, indicating PA was involved in water stress-induced H2O2 and cytosolic free Ca2+ production as well as stress tolerance. Dehydrin genes (Y2SK, Y2K, and SK2) were showed to be highly responsive to exogenous Spd. PA-induced antioxidant defense and dehydrin genes expression could be blocked by the scavenger of H2O2 and the inhibitors of H2O2 generation or Ca2+ channels blockers, a calmodulin antagonist, as well as the inhibitor of CDPK. These findings suggested that PA regulated tolerance to water stress in white clover associated with antioxidant defenses and dehydrins via involvement in the calcium messenger system and H2O2 signaling pathways. PA-induced H2O2 production required Ca2+ release, while PA-induced Ca2+ release was also essential for H2O2 production, suggesting an interaction between PA-induced H2O2 and Ca2+ signaling. PMID:26528187

AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct

PubMed Central

Ahn, Suzie E.; Lee, Sang In; Bazer, Fuller W.; Han, Jae Yong; Song, Gwonhwa

2012-01-01

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor-binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3′-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway. PMID:23145124

Tetrandrine Induces Apoptosis in Human Nasopharyngeal Carcinoma NPC-TW 039 Cells by Endoplasmic Reticulum Stress and Ca2+/Calpain Pathways.

PubMed

Liu, Kuo-Ching; Lin, Ya-Jing; Hsiao, Yung-Ting; Lin, Meng-Liang; Yang, Jiun-Long; Huang, Yi-Ping; Chu, Yung-Lin; Chung, Jing-Gung

2017-11-01

Tetrandrine is an alkaloid extracted from a traditional China medicine plant, and is considered part of food therapy as well. In addition, it has been widely reported to induce apoptotic cell death in many human cancer cells. However, the mechanism of Tetrandrine on human nasopharyngeal carcinoma cells (NPC) is still questioned. In our study, we examined whether Tetrandrine can induce apoptosis of NPC-TW 039 cells. We found that cell morphology was changed after treatment with different concentrations of Tetrandrine. Further, we indicated that the NPC-TW 039 cells viability decreased in a Tetrandrine dose-dependent manner. We also found that tetrandrine induced cell cycle arrest in G 0 /G 1 phase. Tetrandrine induced DNA condensation by DAPI staining as well. In addition, we found that Tetrandrine induced Ca 2+ release in the cytosol. At the same time, endoplasmic reticulum (ER) stress occurred. Then we used western blotting to examine the protein expression which is associated with mitochondria-mediated apoptotic pathways and caspase-dependent pathways. To further examine whether Ca 2+ was released or not with Tetrandrine induced-apoptosis, we used the chelator of Ca 2+ and showed that cell viability increased. At the same time, caspase-3 expression was decreased. Furthermore, confocal microscopy examination revealed that Tetrandrine induced expression of ER stress-related proteins GADD153 and GRP78. Our results indicate that Tetrandrine induces apoptosis through calcium-mediated ER stress and caspase pathway in NPC-TW 039 cells. In conclusion, Tetrandrine may could be used for treatment of human nasopharyngeal carcinoma in future. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The Chemical Molecule B355252 is Neuroprotective in an In Vitro Model of Parkinson's Disease.

PubMed

Gliyazova, Nailya S; Ibeanu, Gordon C

2016-10-01

6-Hydroxydopamine (6-OHDA) is a neurotoxin frequently used to create in vitro and in vivo experimental models of Parkinson's disease (PD), a chronic neurodegenerative disorder largely resulting from damage to the nigrostriatal dopaminergic pathway. No effective drugs or therapies have been developed for this devastating disorder, and current regimens of symptomatic therapeutics only alleviate symptoms temporarily. Therefore, effective treatments that reverse or cure this disorder are urgently needed. The aim of the study described in this report was to investigate the therapeutic impact of B355252, an aryl thiophene sulfonamide chemical entity, in the widely recognized in vitro model of PD, and to characterize the molecular signaling pathways. We show here that 6-OHDA-induced cell death in HT22, a murine neuronal cell model, through a pathway that involves the mitochondria by increasing the levels of reactive oxygen species (ROS), raising intracellular calcium ([Ca(2+)]i), enhancing the release of cytochrome c to the cytosol, and promoting activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway. More importantly, we found that B355252 protected HT22 neurons against 6-OHDA toxin-induced neuronal cell death by significant attenuation of ROS production, blocking of mitochondrial depolarization, inhibition of cytochrome c release, sequestration of [Ca(2+)]i, modulation of JNK cascade, and strong inhibition of caspase 3/7 cleavage. Overall, this study demonstrates that death of neurons under toxic conditions characteristic of PD can be efficiently halted by B355252 and suggests that further development of the molecule could be potentially beneficial as a therapeutic prevention or treatment option for PD.

Calcium-induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization

PubMed Central

Cadetti, Lucia; Bryson, Eric J.; Ciccone, Cory A.; Rabl, Katalin; Thoreson, Wallace B.

2008-01-01

We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (−70 to −10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 μM) had no significant effect on rod calcium current (Ica) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 μM ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 μM) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods. PMID:16819987

Targeting Chronic and Neuropathic Pain: The N-type Calcium Channel Comes of Age

PubMed Central

Snutch, Terrance P.

2005-01-01

Summary: The rapid entry of calcium into cells through activation of voltage-gated calcium channels directly affects membrane potential and contributes to electrical excitability, repetitive firing patterns, excitation-contraction coupling, and gene expression. At presynaptic nerve terminals, calcium entry is the initial trigger mediating the release of neurotransmitters via the calcium-dependent fusion of synaptic vesicles and involves interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex of synaptic release proteins. Physiological factors or drugs that affect either presynaptic calcium channel activity or the efficacy of calcium-dependent vesicle fusion have dramatic consequences on synaptic transmission, including that mediating pain signaling. The N-type calcium channel exhibits a number of characteristics that make it an attractive target for therapeutic intervention concerning chronic and neuropathic pain conditions. Within the past year, both U.S. and European regulatory agencies have approved the use of the cationic peptide Prialt for the treatment of intractable pain. Prialt is the first N-type calcium channel blocker approved for clinical use and represents the first new proven mechanism of action for chronic pain intervention in many years. The present review discusses the rationale behind targeting the N-type calcium channel, some of the limitations confronting the widespread clinical application of Prialt, and outlines possible strategies to improve upon Prialt's relatively narrow therapeutic window. PMID:16489373

Reverse micelle-mediated synthesis of calcium phosphate nanocarriers for controlled release of bovine serum albumin.

PubMed

Dasgupta, Sudip; Bandyopadhyay, Amit; Bose, Susmita

2009-10-01

Calcium phosphate (CaP) nanoparticles with a calcium to phosphorus (Ca:P) molar ratio of 1.5:1 were synthesized using reverse microemulsion. Ca(NO(3))(2).4H(2)O and H(3)PO(4) were used as the aqueous phase, cyclohexane as the organic phase and poly(oxyethylene)(12) nonylphenol ether (NP-12) as the surfactant. Depending on the calcination temperature between 600 and 800 degrees C, CaP nanoparticle showed different phases of calcium-deficient hydroxyapatite (CDHA) and beta-tricalcium phosphate (beta-TCP), particle size between 48 and 69 nm, and a BET specific average surface area between 73 and 57 m(2)g(-1). Bovine serum albumin (BSA) was used as a model protein to study loading and release behavior. The adsorptive property of BSA was investigated by the change in BET surface area of these nanoparticles and the pH of the suspension. At pH 7.5, the maximum amount of BSA was adsorbed onto CaP nanoparticle. The release kinetics of BSA showed a gradual time-dependent increase in pH 4.0 and 6.0 buffer solutions. However, the amount of protein released was significantly smaller at pH 7.2. The BSA release rate also varied depending on the presence of different phases of CaPs in the system, beta-TCP or CDHA. These results suggest that the BSA protein release rate can be controlled by changing the particle size, surface area and phase composition of the CaP nanocarriers.

Stimulation of fibroblast growth factor 23 by metabolic acidosis requires osteoblastic intracellular calcium signaling and prostaglandin synthesis.

PubMed

Krieger, Nancy S; Bushinsky, David A

2017-10-01

Serum fibroblast growth factor 23 (FGF23) increases progressively in chronic kidney disease (CKD) and is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the factors regulating its production are not clear. Patients with CKD have decreased renal acid excretion leading to metabolic acidosis (MET). During MET, acid is buffered by bone with release of mineral calcium (Ca) and phosphate (P). MET increases intracellular Ca signaling and cyclooxygenase 2 (COX2)-induced prostaglandin production in the osteoblast, leading to decreased bone formation and increased bone resorption. We found that MET directly stimulates FGF23 in mouse bone organ cultures and primary osteoblasts. We hypothesized that MET increases FGF23 through similar pathways that lead to bone resorption. Neonatal mouse calvariae were incubated in neutral (NTL, pH = 7.44, Pco 2 = 38 mmHg, [HCO 3 - ] = 27 mM) or acid (MET, pH = 7.18, Pco 2 = 37 mmHg, [HCO 3 - ] = 13 mM) medium without or with 2-APB (50 μM), an inhibitor of intracellular Ca signaling or NS-398 (1 μM), an inhibitor of COX2. Each agent significantly inhibited MET stimulation of medium FGF23 protein and calvarial FGF23 RNA as well as bone resorption at 48 h. To exclude the potential contribution of MET-induced bone P release, we utilized primary calvarial osteoblasts. In these cells each agent inhibited MET stimulation of FGF23 RNA expression at 6 h. Thus stimulation of FGF23 by MET in mouse osteoblasts utilizes the same initial signaling pathways as MET-induced bone resorption. Therapeutic interventions directed toward correction of MET, especially in CKD, have the potential to not only prevent bone resorption but also lower FGF23 and perhaps decrease mortality. Copyright © 2017 the American Physiological Society.

Na+/H+ exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous β-globin knockout thalassemic mice.

PubMed

Charoenphandhu, Narattaphol; Kraidith, Kamonshanok; Lertsuwan, Kornkamon; Sripong, Chanakarn; Suntornsaratoon, Panan; Svasti, Saovaros; Krishnamra, Nateetip; Wongdee, Kannikar

2017-03-01

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous β-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe 2+ and H + cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na + /H + exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na + /Ca 2+ exchanger (NCX1) and plasma membrane Ca 2+ -ATPase (PMCA 1b ), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in β-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

Optimization of Adhesive Pastes for Dental Caries Prevention.

PubMed

Sodata, Patteera; Juntavee, Apa; Juntavee, Niwut; Peerapattana, Jomjai

2017-11-01

Dental caries prevention products available on the market contain only remineralizing agents or antibacterial agents. This study aimed to develop adhesive pastes containing calcium phosphate and α-mangostin for dental caries prevention using the optimization technique. Calcium phosphate was used as a remineralizing agent, and extracted α-mangostin was used as an antibacterial agent. The effect of the independent variables, which were fumed silica, Eudragit ® EPO, polyethylene glycol, and ethyl alcohol, on the responses was investigated. The drying time, erosion rate, calcium release rate, and α-mangostin release rate were established as the measured responses. An equation and a model of the relationship were constructed. An optimal formulation was obtained, and its effect on dental caries prevention was investigated using the pH-cycling model. The quadratic equation revealed that the drying time, calcium release rate, and α-mangostin release rate tended to decrease when increasing the fumed silica and decreasing other factors. The erosion rate tended to increase when decreasing Eudragit ® EPO and increasing other factors. The observed responses of the optimal adhesive pastes were not significantly different from the predicted responses. This result demonstrated that optimization is an efficient technique in the formulation development of the adhesive pastes. In addition, the optimal adhesive pastes could enhance acid resistance activity to the tooth enamel.

Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

NASA Technical Reports Server (NTRS)

Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.

2002-01-01

Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

PubMed Central

Miao, Yi-Liang; Williams, Carmen J.

2012-01-01

Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

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

PubMed

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.

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

PubMed

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.

The Calcium-binding Proteins S100A8 and S100A9 Initiate the Early Inflammatory Program in Injured Peripheral Nerves*

PubMed Central

Chernov, Andrei V.; Dolkas, Jennifer; Hoang, Khang; Angert, Mila; Srikrishna, Geetha; Vogl, Thomas; Baranovskaya, Svetlana; Strongin, Alex Y.; Shubayev, Veronica I.

2015-01-01

To shed light on the early immune response processes in severed peripheral nerves, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P, regenerating) and distal (D, degenerating) nerve stumps on day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, IL, TNF, MHC, immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase, tissue inhibitor of metalloproteinase, and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerves. S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that, following peripheral nerve injury, an immediate acute immune response occurs both distal and proximal to the lesion site and that the rapid transcriptional activation of the S100a8 and S100a9 genes results in S100A8/A9 hetero- and homodimers, which stimulate the release of chemokines and cytokines by activated Schwann cells and generate the initial chemotactic gradient that guides the transmigration of hematogenous immune cells into the injured nerve. PMID:25792748

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

PubMed

Yan, Xiaodong; Liu, Juanfang; Zhang, Zhengping; Li, Wenhao; Sun, Siguo; Zhao, Jian; Dong, Xin; Qian, Jixian; Sun, Honghui

2017-01-01

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca 2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca 2+ ] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca 2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca 2+ ) stores. Blockade of Ca 2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca 2+ -ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca 2+ -dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

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

PubMed

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.

Seagrass-Mediated Phosphorus and Iron Solubilization in Tropical Sediments

PubMed Central

2017-01-01

Tropical seagrasses are nutrient-limited owing to the strong phosphorus fixation capacity of carbonate-rich sediments, yet they form densely vegetated, multispecies meadows in oligotrophic tropical waters. Using a novel combination of high-resolution, two-dimensional chemical imaging of O2, pH, iron, sulfide, calcium, and phosphorus, we found that tropical seagrasses are able to mobilize the essential nutrients iron and phosphorus in their rhizosphere via multiple biogeochemical pathways. We show that tropical seagrasses mobilize phosphorus and iron within their rhizosphere via plant-induced local acidification, leading to dissolution of carbonates and release of phosphate, and via local stimulation of microbial sulfide production, causing reduction of insoluble Fe(III) oxyhydroxides to dissolved Fe(II) with concomitant phosphate release into the rhizosphere porewater. These nutrient mobilization mechanisms have a direct link to seagrass-derived radial O2 loss and secretion of dissolved organic carbon from the below-ground tissue into the rhizosphere. Our demonstration of seagrass-derived rhizospheric phosphorus and iron mobilization explains why seagrasses are widely distributed in oligotrophic tropical waters. PMID:29149570

Extracellular Ca2+ Acts as a Mediator of Communication from Neurons to Glia

PubMed Central

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

2013-01-01

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 ([Ca2+]e) that occur during synaptic transmission might mediate signaling from neurons to glia. Using noninvasive photolysis of the photolabile Ca2+ buffer diazo-2 {N-[2-[2-[2-[bis(carboxymethyl)amino]-5-(diazoacetyl)phenoxy]ethoxy]-4-methylphenyl]-N-(carboxymethyl)-, tetrapotassium salt} to reduce [Ca2+]e or caged glutamate to simulate glutamatergic transmission, we found that a local decline in extracellular Ca2+ triggered astrocytic adenosine triphosphate (ATP) release and astrocytic Ca2+ 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 [Ca2+]e may provide a negative feedback mechanism that potentiates inhibitory transmission in response to local hyperexcitability. PMID:22275221

Neuronal calcium sensor-1 deletion in the mouse decreases motivation and dopamine release in the nucleus accumbens.

PubMed

Ng, Enoch; Varaschin, Rafael K; Su, Ping; Browne, Caleb J; Hermainski, Joanna; Le Foll, Bernard; Pongs, Olaf; Liu, Fang; Trudeau, Louis-Eric; Roder, John C; Wong, Albert H C

2016-03-15

Calcium sensors detect intracellular calcium changes and interact with downstream targets to regulate many functions. Neuronal Calcium Sensor-1 (NCS-1) or Frequenin is widely expressed in the nervous system, and involved in neurotransmission, synaptic plasticity and learning. NCS-1 interacts with and regulates dopamine D2 receptor (D2R) internalization and is implicated in disorders like schizophrenia and substance abuse. However, the role of NCS-1 in behaviors dependent on dopamine signaling in the striatum, where D2R is most highly expressed, is unknown. We show that Ncs-1 deletion in the mouse decreases willingness to work for food. Moreover, Ncs-1 knockout mice have significantly lower activity-dependent dopamine release in the nucleus accumbens core in acute slice recordings. In contrast, food preference, responding for conditioned reinforcement, ability to represent changes in reward value, and locomotor response to amphetamine are not impaired. These studies identify novel roles for NCS-1 in regulating activity-dependent striatal dopamine release and aspects of motivated behavior. Copyright © 2015 Elsevier B.V. All rights reserved.

HIV-1-associated inflammation and antiretroviral therapy regulate astrocyte endoplasmic reticulum stress responses.

PubMed

Nooka, Shruthi; Ghorpade, Anuja

2017-01-01

Antiretroviral (ARV) therapy (ART) has effectively suppressed the incidence of human immunodeficiency virus (HIV)-associated dementia in HIV-1 positive individuals. However, the prevalence of more subtle forms of neurocognitive dysfunction continues to escalate. Recently, endoplasmic reticulum (ER) stress has been linked to many neurological diseases; yet, its role in HIV/neuroAIDS remains largely unexplored. Furthermore, upregulation of astrocyte elevated gene-1 ( AEG-1 ), a novel HIV-1 inducible gene, along with ER stress markers in a Huntington's disease model, suggests a possible role in HIV-associated ER stress. The current study is focused on unfolded protein responses (UPRs) and AEG-1 regulation in primary human astrocytes exposed to HIV-associated neurocognitive disorders (HAND)-relevant stimuli (HIV-1 virions, inflammation and ARV drugs). Interleukin (IL)-1 β and the nucleoside reverse transcriptase inhibitor abacavir upregulated expression of ER stress markers in human astrocytes, including binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), and calnexin. In addition, IL-1 β activated all three well-known UPR pathways: protein kinase RNA-like ER kinase (PERK); activating transcription factor 6 (ATF-6); and inositol-requiring enzyme 1 α (IRE1 α ). AEG-1 upregulation correlated to ER stress and demonstrated astrocyte AEG-1 interaction with the calcium-binding chaperone, calnexin. IL-1 β and abacavir enhanced intracellular calcium signaling in astrocytes in the absence of extracellular calcium, illustrating ER-associated calcium release. Alternatively, calcium evoked in response to HAND-relevant stimuli led to mitochondrial permeability transition pore (mPTP) opening in human astrocytes. Importantly, IL-1 β - and abacavir-induced UPR and mPTP opening were inhibited by the intracellular calcium chelation, indicating the critical role of calcium signaling in HAND-relevant ER stress in astrocytes. In summary, our study highlights that ARV drugs and IL-1 β induced UPR, AEG-1 expression, intracellular calcium, and mitochondrial depolarization in astrocytes. This study uncovers astrocyte ER stress as a novel therapeutic target in the management of HIV-1-associated neurotoxicity and possibly in the treatment of neuroAIDS.

Rechargeable dental adhesive with calcium phosphate nanoparticles for long-term ion release

PubMed Central

Zhang, Ling; Weir, Michael D.; Hack, Gary; Fouad, Ashraf F.; Xu, Hockin H. K.

2015-01-01

Objectives The tooth-resin bond is the weak link of restoration, with secondary caries as a main reason for failure. Calcium phosphate-containing resins are promising for remineralization; however, calcium (Ca) and phosphate (P) ion releases last only a couple of months. The objectives of this study were to develop the first rechargeable CaP bonding agent and investigate the key factors that determine CaP ion recharge and re-release. Methods Nanoparticles of amorphous calcium phosphate (NACP) were synthesized. Pyromellitic glycerol dimethacrylate (PMGDM), ethoxylated bisphenol-A dimethacrylate (EBPADMA), 2-hydroxyethyl methacrylate (HEMA), and bisphenol-A glycidyl dimethacrylate (BisGMA) were used to synthesize three adhesives (denoted PE, PEH and PEHB). NACP were mixed into adhesive at 0–30% by mass. Dentin shear bond strengths were measured. Adhesive specimens were tested for Ca and P initial ion release. Then the ion-exhausted specimens were immersed in Ca and P solution to recharge the specimens, and the recharged specimens were then used to measure ion re-release for 7 days as one cycle. Then these specimens were again recharged and the re-release was measured for 7 days as the second cycle. Three recharge/re-release cycles were tested. Results PEHB had the highest dentin bond strength (p<0.05). Increasing NACP content from 0 to 30% did not affect dentin bond strength (p>0.1), but increased CaP release and re-release (p<0.05). PEHB-NACP had the greatest recharge/re-release, and PE-NACP had the least (p<0.05). Ion release remained high and did not decrease with increasing the number of recharge/re-release cycles (p>0.1). After the third cycle, specimens without further recharge had continuous CaP ion release for 2–3 weeks. Significance Rechargeable CaP bonding agents were developed for the first time to provide long-term Ca and P ions to promote remineralization and reduce caries. Incorporation of NACP into adhesive had no negative effect on dentin bond strength. Increasing NACP filler level increased the ion recharge and re-release capability. The new CaP recharge method and PMGDM-EBPAGMA-NACP composition may have wide application in adhesives, composites and cements, to combat caries and remineralize lesions. PMID:26144190

In Vitro Release and Bioavailability of Silybin from Micelle-Templated Porous Calcium Phosphate Microparticles.

PubMed

Zhu, Yuan; Wang, Miaomiao; Zhang, Ya; Zeng, Jin; Omari-Siaw, E; Yu, Jiangnan; Xu, Ximing

2016-10-01

Developing a promising carrier for the delivery of poorly water-soluble drugs, such as silybin, to improve oral absorption has become a very worthy of consideration. The goal of this study was to prepare a novel porous calcium phosphate microparticle using povidone-mixed micelles as template while evaluating its in vitro and in vivo properties with silybin as a model drug. The particle characterization, in vitro drug release behavior, and pharmacokinetic parameters of the prepared silybin-loaded calcium phosphate microparticle were investigated. The mean particle size was found to be 3.54 ± 0.32 μm with a rough surface porous structure. Additionally, the silybin-loaded calcium phosphate microparticle compared with the free silybin showed a prolonged 72-h release in vitro and a higher C max (418.5 ± 23.7 ng mL(-1)) with 167.5% oral relative bioavailability. A level A in vitro-in vivo correlation (IVIVC), established for the first time, demonstrated an excellent IVIVC of the formulated silybin in oral administration. In conclusion, this povidone-mixed micelle-based microparticle was successfully prepared to enhance the oral bioavailability of silybin. Therefore, application of this novel porous calcium phosphate microparticle holds a significant potential for the development of poorly water-soluble drugs.

Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

PubMed

Santafe, M M; Garcia, N; Lanuza, M A; Tomàs, M; Besalduch, N; Tomàs, J

2009-04-01

We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

[In vitro drug release behavior of carrier made of porous glass ceramics].

PubMed

Wang, De-ping; Huang, Wen-hai; Zhou, Nai

2002-09-01

To conduct the in vitro test on drug release of rifampin encapsulated in a carrier made of porous phosphate glass ceramics and to analyze main factors which affect the drug release rate. A certain quantitative of rifampin was sealed in a hollow cylindrical capsule which consisted of chopped calcium phosphate crystal fiber obtained from glass crystallization. The rifampin concentration was measured in the simulated physiological solution in which the capsule soaked. Rifampin could be released in a constant rate from the porous glass ceramic carrier in a long time. The release rate was dependent on the size of crystal fiber and the wall thickness of the capsule. This kind of calcium phosphate glass ceramics can be a candidate of the carrier materials used as long term drug therapy after osteotomy surgery.

Release of superoxide and change in morphology by neutrophils in response to phorbol esters: antagonism by inhibitors of calcium-binding proteins

PubMed Central

1985-01-01

The ability of phorbol derivatives to function as stimulating agents for superoxide (O2-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate O2- release, while those that are inactive with respect to the kinase have no effect on O2- release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O2- release and morphological changes in these cells. PMID:2993312

Novel cyclodextrin nanosponges for delivery of calcium in hyperphosphatemia.

PubMed

Shende, Pravin; Deshmukh, Kiran; Trotta, Fransesco; Caldera, Fabrizio

2013-11-01

Cyclodextrin nanosponges are solid, porous nanoparticulate three dimensional structures, have been used as delivery system of different drugs. In this work, new cyclodextrin-based nanosponges of calcium carbonate were prepared by polymer condensation method to release the calcium in controlled manner in the treatment of hyperphosphatemia as novel carriers. SEM measurements revealed their roughly spherical shape, porous nature and mean particle size of about 400 nm. Zeta potentials of the nanosponges were sufficiently high to obtain stable formulations. The encapsulation efficiencies of calcium in nanosponge formulations were found to be 81-95%. The moisture contents of the nanosponges were in the range of 0.1-0.7%. The optimized formulation produces enteric and controlled release kinetics of calcium in the management and treatment of hyperphosphatemia. It was also observed that calcium ions bound efficiently to free phosphate in a pH-dependent fashion especially at pH 7. In accelerated stability study no significant changes occurred in physical appearance, size and nature of drug in formulation for 3 months. The results of FTIR and DSC confirmed that calcium carbonate was encapsulated in nanosponges structure. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

PubMed

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 signaling pathway.

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

PubMed

Asmat, Tauseef M; Tenenbaum, Tobias; Jonsson, Ann-Beth; Schwerk, Christian; Schroten, Horst

2014-01-01

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Role of claudins in renal calcium handling.

PubMed

Negri, Armando Luis

2015-01-01

Paracellular channels occurring in tight junctions play a major role in transepithelial ionic flows. This pathway includes a high number of proteins, such as claudins. Within renal epithelium, claudins result in an ionic selectivity in tight junctions. Ascending thick limb of loop of Henle (ATLH) is the most important segment for calcium reabsorption in renal tubules. Its cells create a water-proof barrier, actively transport sodium and chlorine through a transcellular pathway, and provide a paracellular pathway for selective calcium reabsorption. Several studies have led to a model of paracellular channel consisting of various claudins, particularly claudin-16 and 19. Claudin-16 mediates cationic paracellular permeability in ATLH, whereas claudin-19 increases cationic selectivity of claudin-16 by blocking anionic permeability. Recent studies have shown that claudin-14 promoting activity is only located in ATLH. When co-expressed with claudin-16, claudin-14 inhibits the permeability of claudin-16 and reduces paracellular permeability to calcium. Calcium reabsorption process in ATLH is closely regulated by calcium sensor receptor (CaSR), which monitors circulating Ca levels and adjusts renal excretion rate accordingly. Two microRNA, miR-9 and miR-374, are directly regulated by CaSR. Thus, miR-9 and miR-374 suppress mRNA translation for claudin-14 and induce claudin-14 decline. Copyright © 2015 The Author. Published by Elsevier España, S.L.U. All rights reserved.

Priming effect of platelet activating factor on leukotriene C4 from stimulated eosinophils of asthmatic patients.

PubMed Central

Shindo, K.; Koide, K.; Hirai, Y.; Sumitomo, M.; Fukumura, M.

1996-01-01

BACKGROUND: Eosinophils from asthmatic patients are known to release greater amounts of leukotrienes than normal eosinophils when stimulated by the calcium ionophore A23187. The effect of platelet activating factor (PAF) in priming eosinophils was investigated. METHODS: Eosinophils were obtained from 18 asthmatic patients and 18 healthy donors. Cells separated by the Percoll gradients were incubated with PAF (C-18) for 30 minutes and then stimulated with the calcium ionophore A23187 (2.5 microM) for 15 minutes. The amount of leukotriene C4 (LTC4) in supernatants was measured using a combination of high pressure liquid chromatography and radioimmunoassay. RESULTS: The mean (SD) amount of LTC4 released by eosinophils from asthmatic patients upon stimulation with the calcium ionophore A23187 alone was 27.9 (9.9) ng/10(6) cells (n = 6). The amount of LTC4 released following stimulation with the calcium ionophore A23187 after pretreatment with PAF (1, 5, and 10 microM) was 57.2 (8.9), 75.1 (14.3), and 52.6 (10.7) ng/10(6) cells (n = 6), respectively. Trace amounts of LTC4 (0.9 (0.02) ng/10(6) cells, n = 6) were detected in the supernatant of the cells after stimulation by PAF alone (5 microM). The amount of LTC4 released upon stimulation by calcium ionophore A23187 alone in eosinophils from healthy donors was 10.3 (3.7) ng/10(6) cells (n = 4). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with PAF at concentrations of 1, 5, and 10 microM were 11.9 (3.5), 17.8 (5.6), and 12.7 (5.1) ng/10(6) cells (n = 4), respectively. Trace amounts of LTC4 (0.6 (0.02) ng/10(6) cells, n = 4) were detected in the supernatant of the cells upon stimulation with PAF alone (5 microM). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with lyso-PAF at concentrations of 1, 5, and 10 microM (n = 4 or 6) were 30.8 (5.2), 22.9 (5.1), and 27.3 (4.3) ng/10(6) cells (n = 6) from the eosinophils of asthmatic patients and 13.7 (3.3), 15.2 (4.9), and 14.7 (3.8) ng/10(6) cells (n = 4) from the eosinophils of healthy donors. CONCLUSIONS: The results indicated that PAF enhanced LTC4 formation by eosinophils obtained from asthmatic patients stimulated with the calcium ionophore A23187, but not those obtained from normal subjects. PMID:8711647

Induced Plant Defense Responses against Chewing Insects. Ethylene Signaling Reduces Resistance of Arabidopsis against Egyptian Cotton Worm But Not Diamondback Moth1

PubMed Central

Stotz, Henrik U.; Pittendrigh, Barry R.; Kroymann, Jürgen; Weniger, Kerstin; Fritsche, Jacqueline; Bauke, Antje; Mitchell-Olds, Thomas

2000-01-01

The induction of plant defenses by insect feeding is regulated via multiple signaling cascades. One of them, ethylene signaling, increases susceptibility of Arabidopsis to the generalist herbivore Egyptian cotton worm (Spodoptera littoralis; Lepidoptera: Noctuidae). The hookless1 mutation, which affects a downstream component of ethylene signaling, conferred resistance to Egyptian cotton worm as compared with wild-type plants. Likewise, ein2, a mutant in a central component of the ethylene signaling pathway, caused enhanced resistance to Egyptian cotton worm that was similar in magnitude to hookless1. Moreover, pretreatment of plants with ethephon (2-chloroethanephosphonic acid), a chemical that releases ethylene, elevated plant susceptibility to Egyptian cotton worm. By contrast, these mutations in the ethylene-signaling pathway had no detectable effects on diamondback moth (Plutella xylostella) feeding. It is surprising that this is not due to nonactivation of defense signaling, because diamondback moth does induce genes that relate to wound-response pathways. Of these wound-related genes, jasmonic acid regulates a novel β-glucosidase 1 (BGL1), whereas ethylene controls a putative calcium-binding elongation factor hand protein. These results suggest that a specialist insect herbivore triggers general wound-response pathways in Arabidopsis but, unlike a generalist herbivore, does not react to ethylene-mediated physiological changes. PMID:11080278

Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats.

PubMed

Dahan, Diana; Ducret, Thomas; Quignard, Jean-François; Marthan, Roger; Savineau, Jean-Pierre; Estève, Eric

2012-11-01

There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca(2+)](i)). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca(2+)](i) increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca(2+) release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.

Computational study of a calcium release-activated calcium channel

NASA Astrophysics Data System (ADS)

Talukdar, Keka; Shantappa, Anil

2016-05-01

The naturally occurring proteins that form hole in membrane are commonly known as ion channels. They play multiple roles in many important biological processes. Deletion or alteration of these channels often leads to serious problems in the physiological processes as it controls the flow of ions through it. The proper maintenance of the flow of ions, in turn, is required for normal health. Here we have investigated the behavior of a calcium release-activated calcium ion channel with pdb entry 4HKR in Drosophila Melanogaster. The equilibrium energy as well as molecular dynamics simulation is performed first. The protein is subjected to molecular dynamics simulation to find their energy minimized value. Simulation of the protein in the environment of water and ions has given us important results too. The solvation energy is also found using Charmm potential.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

PubMed

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect of CPP-ACP-propolis chewing gum on calcium and phosphate ion release on caries-active subjects’ saliva and the formation of Streptococcus mutans biofilm

NASA Astrophysics Data System (ADS)

Hasnamudhia, F.; Bachtiar, E. W.; Sahlan, M.; Soekanto, S. A.

2017-08-01

The aim of this study was to analyze the effect of CPP-APP and propolis wax if they are combined in a chewing gum formulation, observed from the calcium and phosphate ion level released by CPP-ACP and the emphasis of Streptococcus mutans mass in the biofilm by propolis wax on caries-active subjects’ saliva. Chewing gum simulation was done in vitro on 25 caries-active subjects’ saliva using five concentrations of chewing gum (0% propolis + 0% CPP-ACP, 0% propolis + CPP-ACP, 2% propolis + CPP-ACP, 4% propolis + CPP-ACP, and 6% propolis + CPP-ACP) and was then tested using an atomic absorption spectrophotometer to analyze calcium ion levels, an ultraviolet-visible spectrophotometer to analyze phosphate ion levels, and a biofilm assay using crystal violet to analyze the decline in biofilm mass. After the chewing simulation, calcium ion levels on saliva+gum eluent increased significantly compared to the saliva control, with the highest calcium level released by CPP-ACP + 2% propolis chewing gum. There was an insignificant phosphate level change between the saliva control and saliva+gum eluent. There was also a significant decline of S. mutans biofilm mass in the saliva+gum eluent, mostly by the CPP-ACP chewing gum and CPP-ACP + 6% propolis. The CPP-ACP-propolis chewing gum simulation generated the largest increase in calcium and phosphate ion level and the largest decline in S. mutans biofilm mass.

The effects of substance P on histamine and 5-hydroxytryptamine release in the rat

PubMed Central

Fewtrell, C. M. S.; Foreman, J. C.; Jordan, C. C.; Oehme, P.; Renner, H.; Stewart, J. M.

1982-01-01

1. Substance P (SP) induces histamine release from isolated rat peritoneal mast cells at concentrations of 0·1-10 μM. 2. Inhibitors of glycolysis and oxidative phosphorylation prevent the release of histamine induced by SP. 3. Cells heated to 47 °C for 20 min release histamine when treated with an agent causing cell lysis but fail to release in response to SP. 4. SP does not release histamine by interacting with cell-bound IgE. 5. Histamine release by SP is rapid, with more than 90% of the response occurring within 1 min of the addition of the peptide to mast cells at 37 °C. 6. Substance P, unlike antigen—antibody or compound 48/80, does not show enhanced release of histamine when calcium (0·1-1 mM) is present in the extracellular medium but calcium increases the response to SP when the ion is added after the peptide. Extracellular calcium (0·1-1 mM), magnesium (1-10 mM) and cobalt (0·01-0·1 mM) all inhibit SP-induced histamine release when added before the peptide. Pre-treatment of the cells with EDTA (10 mM) and washing in calcium-free medium inhibits the histamine release induced by SP. 7. Histamine release induced by SP was optimum at an extracellular pH of 7·2. 8. A number of peptides structurally related to SP were examined for histamine-releasing activity. At the concentrations tested, the N-terminal dipeptides Lys-Pro and Arg-Pro, tuftsin, physalaemin, eledoisin, SP3-11, SP4-11 and [p-Glu6, p-amino Phe7]-SP6-11 were all found to be inactive. The relative activities of the other peptides were: [Formula: see text] 9. Rat basophilic leukaemia cells (RBL-2H3) fail to respond to SP at concentrations which activate rat mast cells. Release of 5-hydroxytryptamine by immunological activation of RBL cells is not changed by the presence of SP. 10. The mechanism of action of SP on mast cells and the nature of the SP receptor on mast cells is discussed in relation to SP receptors in other cell types. PMID:6184468

Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation

PubMed Central

Wilson, Calum; Lee, Matthew D.

2016-01-01

Key points The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. Abstract Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction. PMID:27730645

[Study on solid dispersion of precipitated calcium carbonate-based oleanolic acid].

PubMed

Yan, Hong-mei; Zhang, Zhen-hai; Jia, Xiao-bin; Jiang, Yan-rong; Sun, E

2015-05-01

Oleanolic acid-precipitated calcium carbonate solid dispersion was prepared by using solvent evaporation method. The microscopic structure and physicochemical properties of solid dispersion were analyzed using differential scanning calorimetry and scanning electron microscopy (SEM). And its in vitro release also was investigated. The properties of the precipitated calcium carbonate was studied which was as a carrier of oleanolic acid solid dispersion. Differential scanning calorimetry analysis suggested that oleanolic acid may be present in solid dispersion as amorphous substance. The in vitro release determination results of oleanolic acid-precipitated calcium carbonate (1: 5) solid dispersion showed accumulated dissolution rate of.oleanolic acid was up to 90% at 45 min. Accelerating experiment showed that content and in vitro dissolution of oleanolic acid solid dispersion did not change after storing over 6 months. The results indicated that in vitro dissolution of oleanolic acid was improved greatly by the solid dispersion with precipitated calcium carbonate as a carrier. The solid dispersion is a stabilizing system which has actual applied value.

Inorganic nanoparticles as nucleic acid transporters into eukaryotic cells

NASA Astrophysics Data System (ADS)

Amirkhanov, R. N.; Zarytova, V. F.; Zenkova, M. A.

2017-02-01

The review is concerned with inorganic nanoparticles (gold, titanium dioxide, silica, iron oxides, calcium phosphate) used as nucleic acid transporters into mammalian cells. Methods for the synthesis of nanoparticles and approaches to surface modification through covalent or noncovalent attachment of low- or high-molecular-weight compounds are considered. The data available from the literature on biological action of nucleic acids delivered into the cells by nanoparticles and on the effect of nanoparticles and their conjugates and complexes on the cell survival are summarized. Pathways of cellular internalization of nanoparticles and the mechanism of their excretion, as well as the ways of release of nucleic acids from their complexes with nanoparticles after the cellular uptake are described. The bibliography includes 161 references.

Intestinal absorption and renal reabsorption of calcium throughout postnatal development

PubMed Central

Beggs, Megan R

2017-01-01

Calcium is vital for many physiological functions including bone mineralization. Postnatal deposition of calcium into bone is greatest in infancy and continues through childhood and adolescence until peek mineral density is reached in early adulthood. Thereafter, bone mineral density remains static until it eventually declines in later life. A positive calcium balance, i.e. more calcium absorbed than excreted, is crucial to bone deposition during growth and thus to peek bone mineral density. Dietary calcium is absorbed from the intestine into the blood. It is then filtered by the renal glomerulus and either reabsorbed by the tubule or excreted in the urine. Calcium can be (re)absorbed across intestinal and renal epithelia via both transcellular and paracellular pathways. Current evidence suggests that significant intestinal and renal calcium transport changes occur throughout development. However, the molecular details of these alterations are incompletely delineated. Here we first briefly review the current model of calcium transport in the intestine and renal tubule in the adult. Then, we describe what is known with regard to calcium handling through postnatal development, and how alterations may aid in mediating a positive calcium balance. The role of transcellular and paracellular calcium transport pathways and the contribution of specific intestinal and tubular segments vary with age. However, the current literature highlights knowledge gaps in how specifically intestinal and renal calcium (re)absorption occurs early in postnatal development. Future research should clarify the specific changes in calcium transport throughout early postnatal development including mediators of these alterations enabling appropriate bone mineralization. Impact statement This mini review outlines the current state of knowledge pertaining to the molecules and mechanisms maintaining a positive calcium balance throughout postnatal development. This process is essential to achieving optimal bone mineral density in early adulthood, thereby lowering the lifetime risk of osteoporosis. PMID:28346014

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral-derived macroporous constructs

PubMed Central

Klar, Roland M; Duarte, Raquel; Dix-Peek, Therese; Dickens, Caroline; Ferretti, Carlo; Ripamonti, Ugo

2013-01-01

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca++ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca++ activating stem cell differentiation and the induction of bone formation. PMID:24106923

Ca2+ and Mn2+ Influx Through Receptor-Mediated Activation of Nonspecific Cation Channels in Mast Cells

NASA Astrophysics Data System (ADS)

Fasolato, Cristina; Hoth, Markus; Matthews, Gary; Penner, Reinhold

1993-04-01

Whole-cell patch-clamp recordings of membrane currents and Fura-2 measurements of free intracellular calcium concentration ([Ca2+]_i) were used to study calcium influx through receptor-activated cation channels in rat peritoneal mast cells. Cation channels were activated by the secretagogue compound 48/80, whereas a possible concomitant Ca2+ entry through pathways activated by depletion of calcium stores was blocked by dialyzing cells with heparin. Heparin effectively suppressed the transient Ca2+ release induced by 48/80 and abrogated inositol 1,4,5-trisphosphate-induced calcium influx without affecting activation of 50-pS cation channels. There was a clear correlation between changes in [Ca2+]_i and the activity of 50-pS channels. The changes in [Ca2+]_i increased with elevation of extracellular Ca2+. At the same time, inward currents through 50-pS channels were diminished as more Ca2+ permeated. This effect was due to a decrease in slope conductance and a reduction in the open probability of the cation channels. In physiological solutions, 3.6% of the total current was carried by Ca2+. The cation channels were not only permeable to Ca2+ but also to Mn2+, as evidenced by the quench of Fura-2 fluorescence. Mn2+ current through 50-pS channels could not be resolved at the single-channel level. Our results suggest that 50-pS cation channels partially contribute to sustained increases of [Ca2+]_i in mast cells following receptor activation.

From contraction to gene expression: nanojunctions of the sarco/endoplasmic reticulum deliver site- and function-specific calcium signals.

PubMed

Evans, A Mark; Fameli, Nicola; Ogunbayo, Oluseye A; Duan, Jingxian; Navarro-Dorado, Jorge

2016-08-01

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Effect of soluble calcium on the renneting properties of casein micelles as measured by rheology and diffusing wave spectroscopy.

PubMed

Sandra, S; Ho, M; Alexander, M; Corredig, M

2012-01-01

Addition of calcium chloride to milk has positive effects on cheese-making because it decreases coagulation time, creates firmer gels, and increases curd yield. Although addition of calcium chloride is a widely used industrial practice, the effect of soluble calcium on the preliminary stages of gelation is not fully understood. In addition, it is not known whether the manner of addition and equilibration of the soluble calcium would affect the rennetability of the casein micelles. Therefore, the aim of this paper was to study the details of the coagulation behavior of casein micelles in the presence of additional calcium, and to elucidate whether the manner in which this cation is added (directly as calcium chloride or by gradual exchange through dialysis) affects the functionality of the micelles. Calcium was added as CaCl(2) (1 mM final added concentration) directly to skim milk or indirectly using dialysis against 50 volumes of milk. Additional soluble calcium did not affect the primary phase of the renneting reaction, as demonstrated by the analysis of the casein macropeptide (CMP) released in solution; however, it shortened the coagulation time of the micelles and increased the firmness of the gel. The turbidity parameter of samples with or without calcium showed that similar amounts of CMP were needed for particle interactions to commence. However, the amount of CMP released at the point of gelation, as indicated by rheology, was lesser for samples with added calcium, which can be attributed to a greater extent of calcium bridging on the surface or between micelles. The results also showed that the manner in which calcium was presented to the micelles did not influence the mechanism of gelation. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Properties of Ca2+ sparks evoked by action potentials in mouse ventricular myocytes

PubMed Central

Bridge, John H B; Ershler, Philip R; Cannell, Mark B

1999-01-01

Calcium sparks were examined in enzymatically dissociated mouse cardiac ventricular cells using the calcium indicator fluo-3 and confocal microscopy. The properties of the mouse cardiac calcium spark are generally similar to those reported for other species.Examination of the temporal relationship between the action potential and the time course of calcium spark production showed that calcium sparks are more likely to occur during the initial repolarization phase of the action potential. The latency of their occurrence varied by less than 1·4 ms (s.d.) and this low variability may be explained by the interaction of the gating of L-type calcium channels with the changes in driving force for calcium entry during the action potential.When fixed sites within the cell are examined, calcium sparks have relatively constant amplitude but the amplitude of the sparks was variable among sites. The low variability of the amplitude of the calcium sparks suggests that more than one sarcoplasmic reticulum (SR) release channel must be involved in their genesis. Noise analysis (with the assumption of independent gating) suggests that > 18 SR calcium release channels may be involved in the generation of the calcium spark. At a fixed site, the response is close to ‘all-or-none’ behaviour which suggests that calcium sparks are indeed elementary events underlying cardiac excitation-contraction coupling.A method for selecting spark sites for signal averaging is presented which allows the time course of the spark to be examined with high temporal and spatial resolution. Using this method we show the development of the calcium spark at high signal-to-noise levels. PMID:10381593

Vitamin K3 inhibits mouse uterine contraction in vitro via interference with the calcium transfer and the potassium channels.

PubMed

Zhang, Xian-Xia; Lu, Li-Min; Wang, Li

2016-08-05

Previous studies have demonstrated vitamin K3 had a great relief to smooth muscle spastic disorders, but no researches have yet pinpointed its possible anti-contractile activity in the uterus. Here, we evaluated the effect of vitamin K3 on myometrial contractility and explored the possible mechanisms of vitamin K3 action. Myograph apparatus were used to record the changes in contractility of isolated mouse uterine strips in a tissue bath. Uterine strips were exposed to vitamin K3 or vehicle. Vitamin K3 suppressed spontaneous contractions in a concentration dependent manner. It significantly decreased the contractile frequency induced by PGF2ɑ but not their amplitude (expect 58.0 μM). Prior incubation with vitamin K3 reduced the effectiveness of PGF2ɑ-induced contraction. The antispasmodic effect of vitamin K3 was also sensitive to potassium channel blockers, such as tetraethylammonium, 4-aminopyridine, iberiotoxin) but not to the nitric oxide related pathway blockers. High concentrations (29.0, 58.0 μM) of vitamin K3 weakened the Ca(2+) dose response and inhibited phase 1 contraction (intracellular stored calcium release). These dates suggest that vitamin K3 specifically suppresses myometrial contractility by affecting calcium and potassium channels; thus, this approach has potential therapy for uterine contractile activity related disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Eluted zinc ions stimulate osteoblast differentiation and mineralization in human dental pulp stem cells for bone tissue engineering.

PubMed

Yusa, Kazuyuki; Yamamoto, Osamu; Iino, Mitsuyoshi; Takano, Hiroshi; Fukuda, Masayuki; Qiao, Zhiwei; Sugiyama, Toshihiro

2016-11-01

Zinc is an essential element for proliferation, differentiation and survival in various cell types. In a previous study, we found that zinc ions released from zinc-modified titanium surfaces (eluted zinc ions; EZ) stimulate cell viability, osteoblast marker gene expression and calcium deposition in human bone marrow-derived mesenchymal cells (hBMCs). The aim of the present study was to investigate the effects of EZ on osteoblast differentiation among dental pulp stem cells (DPSCs) in vitro. In this study, we evaluated the effects of EZ on osteogenesis in DPSCs. Osteoblast and osteoclast marker gene expression was evaluated by real-time PCR. We also evaluated alkaline phosphatase (ALP) staining and calcium deposition. We found that EZ stimulated osteoblast marker gene (type I collagen, alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2) expression, vascular endothelial growth factor A (VEGF-A), and TGF-beta signaling pathway-related gene expression after 7days of incubation. Osteoclastogenesis occurs in a receptor for activated nuclear-factor kappa B ligand (RANKL)/osteoprotegerin (OPG)-independent manner. Real-time PCR analysis revealed that EZ did not affect RANKL or OPG mRNA expression. It was also revealed that EZ induced alkaline phosphatase (ALP) staining and calcium deposition in DPSCs. Collectively, these results demonstrate the potential for clinical application to prospective treatment of bone diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metabotropic and ionotropic glutamate receptors regulate calcium channel currents in salamander retinal ganglion cells

PubMed Central

Shen, Wen; Slaughter, Malcolm M

1998-01-01

Glutamate suppressed high-voltage-activated barium currents (IBa,HVA) in tiger salamander retinal ganglion cells. Both ionotropic (iGluR) and metabotropic (mGluR) receptors contributed to this calcium channel inhibition. Trans-ACPD (1-aminocyclopentane-trans-1S,3R-dicarboxylic acid), a broad-spectrum metabotropic glutamate receptor agonist, suppressed a dihydropyridine-sensitive barium current. Kainate, an ionotropic glutamate receptor agonist, reduced an ω-conotoxin GVIA-sensitive current. The relative effectiveness of selective agonists indicated that the predominant metabotropic receptor was the L-2-amino-4-phosphonobutyrate (l-AP4)-sensitive, group III receptor. This receptor reversed the action of forskolin, but this was not responsible for calcium channel suppression. l-AP4 raised internal calcium concentration. Antagonists of phospholipase C, inositol trisphosphate (IP3) receptors and ryanodine receptors inhibited the action of metabotropic agonists, indicating that group III receptor transduction was linked to this pathway. The action of kainate was partially suppressed by BAPTA, by calmodulin antagonists and by blockers of calmodulin-dependent phosphatase. Suppression by kainate of the calcium channel current was more rapid when calcium was the charge carrier, instead of barium. The results indicate that calcium influx through kainate-sensitive glutamate receptors can activate calmodulin, which stimulates phosphatases that may directly suppress voltage-sensitive calcium channels. Thus, ionotropic and metabotropic glutamate receptors inhibit distinct calcium channels. They could act synergistically, since both increase internal calcium. These pathways provide negative feedback that can reduce calcium influx when ganglion cells are depolarized. PMID:9660896

Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

PubMed Central

2014-01-01

Background Prolonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways. Results Using in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3 channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode. Conclusions Our findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions. PMID:24965271

Synaptic transmission at the endbulb of Held deteriorates during age‐related hearing loss

PubMed Central

Manis, Paul B.

2016-01-01

Key points Synaptic transmission at the endbulb of Held was assessed by whole‐cell patch clamp recordings from auditory neurons in mature (2–4 months) and aged (20–26 months) mice.Synaptic transmission is degraded in aged mice, which may contribute to the decline in neural processing of the central auditory system during age‐related hearing loss.The changes in synaptic transmission in aged mice can be partially rescued by improving calcium buffering, or decreasing action potential‐evoked calcium influx.These experiments suggest potential mechanisms, such as regulating intraterminal calcium, that could be manipulated to improve the fidelity of transmission at the aged endbulb of Held. Abstract Age‐related hearing loss (ARHL) is associated with changes to the auditory periphery that raise sensory thresholds and alter coding, and is accompanied by alterations in excitatory and inhibitory synaptic transmission, and intrinsic excitability in the circuits of the central auditory system. However, it remains unclear how synaptic transmission changes at the first central auditory synapses during ARHL. Using mature (2–4 months) and old (20–26 months) CBA/CaJ mice, we studied synaptic transmission at the endbulb of Held. Mature and old mice showed no difference in either spontaneous quantal synaptic transmission or low frequency evoked synaptic transmission at the endbulb of Held. However, when challenged with sustained high frequency stimulation, synapses in old mice exhibited increased asynchronous transmitter release and reduced synchronous release. This suggests that the transmission of temporally precise information is degraded at the endbulb during ARHL. Increasing intraterminal calcium buffering with EGTA‐AM or decreasing calcium influx with ω‐agatoxin IVA decreased the amount of asynchronous release and restored synchronous release in old mice. In addition, recovery from depression following high frequency trains was faster in old mice, but was restored to a normal time course by EGTA‐AM treatment. These results suggest that intraterminal calcium in old endbulbs may rise to abnormally high levels during high rates of auditory nerve firing, or that calcium‐dependent processes involved in release are altered with age. These observations suggest that ARHL is associated with a decrease in temporal precision of synaptic release at the first central auditory synapse, which may contribute to perceptual deficits in hearing. PMID:27618790

Single-Cell Phenotypic Characterization of Human Pituitary GHomas and Non-Functioning Adenomas Based on Hormone Content and Calcium Responses to Hypothalamic Releasing Hormones

PubMed Central

Senovilla, Laura; Núñez, Lucía; de Campos, José María; de Luis, Daniel A.; Romero, Enrique; García-Sancho, Javier; Villalobos, Carlos

2015-01-01

Human pituitary tumors are generally benign adenomas causing considerable morbidity due to excess hormone secretion, hypopituitarism, and other tumor mass effects. Pituitary tumors are highly heterogeneous and difficult to type, often containing mixed cell phenotypes. We have used calcium imaging followed by multiple immunocytochemistry to type growth hormone secreting (GHomas) and non-functioning pituitary adenomas (NFPAs). Individual cells were typed for stored hormones and calcium responses to classic hypothalamic releasing hormones (HRHs). We found that GHomas contained growth hormone cells either lacking responses to HRHs or responding to all four HRHs. However, most GHoma cells were polyhormonal cells responsive to both thyrotropin-releasing hormone (TRH) and GH-releasing hormone. NFPAs were also highly heterogeneous. Some of them contained ACTH cells lacking responses to HRHs or polyhormonal gonadotropes responsive to LHRH and TRH. However, most NFPAs were made of cells storing no hormone and responded only to TRH. These results may provide new insights on the ontogeny of GHomas and NFPAs. PMID:26106585

Atorvastatin Calcium Inhibits Phenotypic Modulation of PDGF-BB-Induced VSMCs via Down-Regulation the Akt Signaling Pathway

PubMed Central

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. PMID:25874930

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

PubMed Central

Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

2008-01-01

Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum.

PubMed

Paydar, Mehrak Javadi; Pousti, Abbas; Farsam, Hasan; Amanlou, Massoud; Mehr, Shahram Ejtemaei; Dehpour, Ahmad Reza

2005-11-01

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.

Carbon-Based Solid-State Calcium Ion-Selective Microelectrode and Scanning Electrochemical Microscopy: A Quantitative Study of pH-Dependent Release of Calcium Ions from Bioactive Glass.

PubMed

Ummadi, Jyothir Ganesh; Downs, Corey J; Joshi, Vrushali S; Ferracane, Jack L; Koley, Dipankar

2016-03-15

Solid-state ion-selective electrodes are used as scanning electrochemical microscope (SECM) probes because of their inherent fast response time and ease of miniaturization. In this study, we report the development of a solid-state, low-poly(vinyl chloride), carbon-based calcium ion-selective microelectrode (Ca(2+)-ISME), 25 μm in diameter, capable of performing an amperometric approach curve and serving as a potentiometric sensor. The Ca(2+)-ISME has a broad linear response range of 5 μM to 200 mM with a near Nernstian slope of 28 mV/log[a(Ca(2+))]. The calculated detection limit for Ca(2+)-ISME is 1 μM. The selectivity coefficients of this Ca(2+)-ISME are log K(Ca(2+),A) = -5.88, -5.54, and -6.31 for Mg(2+), Na(+), and K(+), respectively. We used this new type of Ca(2+)-ISME as an SECM probe to quantitatively map the chemical microenvironment produced by a model substrate, bioactive glass (BAG). In acidic conditions (pH 4.5), BAG was found to increase the calcium ion concentration from 0.7 mM ([Ca(2+)] in artificial saliva) to 1.4 mM at 20 μm above the surface. In addition, a solid-state dual SECM pH probe was used to correlate the release of calcium ions with the change in local pH. Three-dimensional pH and calcium ion distribution mapping were also obtained by using these solid-state probes. The quantitative mapping of pH and Ca(2+) above the BAG elucidates the effectiveness of BAG in neutralizing and releasing calcium ions in acidic conditions.

Expression of the P/Q (Cav2.1) calcium channel in nodose sensory neurons and arterial baroreceptors.

PubMed

Tatalovic, Milos; Glazebrook, Patricia A; Kunze, Diana L

2012-06-27

The predominant calcium current in nodose sensory neurons, including the subpopulation of baroreceptor neurons, is the N-type channel, Cav2.2. It is also the primary calcium channel responsible for transmitter release at their presynaptic terminals in the nucleus of the solitary tract in the brainstem. The P/Q channel, Cav2.1, the other major calcium channel responsible for transmitter release at mammalian synapses, represents only 15-20% of total calcium current in the general population of sensory neurons and makes a minor contribution to transmitter release at the presynaptic terminal. In the present study we identified a subpopulation of the largest nodose neurons (capacitance>50pF) in which, surprisingly, Cav2.1 represents over 50% of the total calcium current, differing from the remainder of the population. Consistent with these electrophysiological data, anti-Cav2.1 antibody labeling was more membrane delimited in a subgroup of the large neurons in slices of nodose ganglia. Data reported in other synapses in the central nervous system assign different roles in synaptic information transfer to the P/Q-type versus N-type calcium channels. The study raises the possibility that the P/Q channel which has been associated with high fidelity transmission at other central synapses serves a similar function in this group of large myelinated sensory afferents, including arterial baroreceptors where a high frequency regular discharge pattern signals the pressure pulse. This contrasts to the irregular lower frequency discharge of the unmyelinated fibers that make up the majority of the sensory population and that utilize the N-type channel in synaptic transmission. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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.

Calcium channel dynamics limit synaptic release in response to prosthetic stimulation with sinusoidal waveforms

PubMed Central

Freeman, Daniel K.; Jeng, Jed S.; Kelly, Shawn K.; Hartveit, Espen; Fried, Shelley I.

2011-01-01

Extracellular electric stimulation with sinusoidal waveforms has been shown to allow preferential activation of individual types of retinal neurons by varying stimulus frequency. It is important to understand the mechanisms underlying this frequency dependence as a step towards improving methods of preferential activation. In order to elucidate these mechanisms, we implemented a morphologically realistic model of a retinal bipolar cell and measured the response to extracellular stimulation with sinusoidal waveforms. We compared the frequency response of a passive membrane model to the kinetics of voltage-gated calcium channels that mediate synaptic release. The passive electrical properties of the membrane exhibited lowpass filtering with a relatively high cutoff frequency (nominal value = 717 Hz). This cutoff frequency was dependent on intra-axonal resistance, with shorter and wider axons yielding higher cutoff frequencies. However, we found that the cutoff frequency of bipolar cell synaptic release was primarily limited by the relatively slow opening kinetics of Land T-type calcium channels. The cutoff frequency of calcium currents depended nonlinearly on stimulus amplitude, but remained lower than the cutoff frequency of the passive membrane model for a large range of membrane potential fluctuations. These results suggest that while it may be possible to modulate the membrane potential of bipolar cells over a wide range of stimulus frequencies, synaptic release will only be initiated at the lower end of this range. PMID:21628768

Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes

PubMed Central

1983-01-01

Unfertilized eggs of the medaka fish (Oryzias latipes) were injected with pH-buffered calcium buffers. Medaka egg activation is accompanied by a transient increase in cytoplasmic free calcium (Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds, 1978, J. Cell Biol., 76:448-466). The calcium buffer injections demonstrated that (a) the threshold free calcium required to elicit the calcium transient and activate the egg is between 1.7 and 5.1 microM at pH 7.0, well below the 30 microM reached during the transient, and (b) buffers which hold free calcium below threshold prevent activation of the buffered region in subsequently fertilized eggs. Therefore an increase in free calcium is necessary and sufficient to elicit the calcium transient, and the calcium transient is necessary to activate the egg. Further, these results are additional proof that the calcium transient is initiated and propagated through the cytoplasm by a mechanism of calcium- stimulated calcium release. Finally, a normal calcium transient must propagate through the entire cytoplasm to ensure normal development. Unfertilized eggs were injected with pH buffers to produce short-term, localized changes in cytoplasmic pH. The eggs were then fertilized at various times after injection. In other experiments, unfertilized and fertilized eggs were exposed to media containing either NH4Cl or CO2 to produce longer term, global changes in cytoplasmic pH. These treatments neither activated the eggs nor interfered with the normal development of fertilized eggs, suggesting that even if a natural change in cytoplasmic pH is induced by activation, it has no role in medaka egg development. The injected pH buffers altered the rate of propagation of the calcium transient through the cytoplasm, suggesting that the threshold free calcium required to trigger calcium-stimulated calcium release might be pH dependent. The results of injection of pH-buffered calcium buffers support this conjecture: for a tenfold increase in hydrogen ion concentration, free calcium must also be raised tenfold to elicit the calcium transient. PMID:6411737

Calcium in the control of renin release.

PubMed

Park, C S; Malvin, R L

1978-07-01

The effect of Ca concentrations in the incubation medium and of estimated intracellular Ca concentrations on renin release was examined with use of pig renal cortical slices. In addition, the Ca requirement for the epinephrine stimulatory effect and for the ouabain inhibitory action on renin release was also tested. In mediums containing 5.9 mM K, variations in Ca concentration had no effect on renin release. In contrast, when the K concentration was 59 mM, a significant inhibition of renin release was attained with all concentrations of calcium. The inhibition of renin release in high K mediums by Ca was attributed to an increase in the intracellular Ca concentration. In addition, both the stimulatory effect of epinephrine and the inhibitory effect of ouabain on renin release required Ca in the medium. These results support the hypothesis that the control of renin secretion is mediated, in part, by changes in the intracellular concentration of Ca, most likely in the juxtaglomerular cells.

Probing the structure of the conduction pathway of the sheep cardiac sarcoplasmic reticulum calcium-release channel with permeant and impermeant organic cations

PubMed Central

1993-01-01

The sarcoplasmic reticulum Ca(2+)-release channel plays a central role in cardiac muscle function by providing a ligand-regulated pathway for the release of sequestered Ca2+ to initiate contraction following cell excitation. The efficiency of the channel as a Ca(2+)-release pathway will be influenced by both gating and conductance properties of the system. In the past we have investigated conduction and discrimination of inorganic mono- and divalent cations with the aim of describing the mechanisms governing ion handling in the channel (Tinker, A., A.R. G. Lindsay, and A.J. Williams. 1992. Journal of General Physiology. 100:495-517.). In the present study, we have used permeant and impermeant organic cations to provide additional information on structural features of the conduction pathway. The use of permeant organic cations in biological channels to explore structural motifs underlying selectivity has been an important tool for the electrophysiologist. We have examined the conduction properties of a series of monovalent organic cations of varying size in the purified sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel. Relative permeability, determined from the reversal potential measured under bi- ionic conditions with 210-mM test cation at the cytoplasmic face of the channel and 210 mM K+ at the luminal, was related inversely to the minimum circular cation radius. The reversal potential was concentration-independent. The excluded area hypothesis, with and without a term for solute-wall friction, described the data well and gave a lower estimate for minimum pore radius of 3.3-3.5 A. Blocking studies with the impermeant charged derivative of triethylamine reveal that this narrowing occurs over the first 10-20% of the voltage drop when crossing from the lumen of the SR to the cytoplasm. Single-channel conductances were measured in symmetrical 210 mM salt. Factors other than relative permeability determine conductance as ions with similar relative permeability can have widely varying single-channel conductance. Permeant ions, such as the charged derivatives of trimethylamine and diethylmethylamine, can also inhibit K+ current. The reduction in relative conductance with increasing concentrations of these two ions at a holding potential of 60 mV was described by a rectangular hyperbola and revealed higher affinity binding for diethylmethylamine as compared to trimethylamine. It was possible to describe the complex permeation properties of these two ions using a single-ion four barrier, three binding site Eyring rate theory model. In conclusion, these studies reveal that the cardiac Ca(2+)-release channel has a selectivity filter of approximately 3.5-A radius located at the luminal face of the protein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8133241

Novel rechargeable calcium phosphate nanoparticle-containing orthodontic cement.

PubMed

Xie, Xian-Ju; Xing, Dan; Wang, Lin; Zhou, Han; Weir, Michael D; Bai, Yu-Xing; Xu, Hockin Hk

2017-03-01

White spot lesions (WSLs), due to enamel demineralization, occur frequently in orthodontic treatment. We recently developed a novel rechargeable dental composite containing nanoparticles of amorphous calcium phosphate (NACP) with long-term calcium (Ca) and phosphate (P) ion release and caries-inhibiting capability. The objectives of this study were to develop the first NACP-rechargeable orthodontic cement and investigate the effects of recharge duration and frequency on the efficacy of ion re-release. The rechargeable cement consisted of pyromellitic glycerol dimethacrylate (PMGDM) and ethoxylated bisphenol A dimethacrylate (EBPADMA). NACP was mixed into the resin at 40% by mass. Specimens were tested for orthodontic bracket shear bond strength (SBS) to enamel, Ca and P ion initial release, recharge and re-release. The new orthodontic cement exhibited an SBS similar to commercial orthodontic cement without CaP release (P>0.1). Specimens after one recharge treatment (e.g., 1 min immersion in recharge solution repeating three times in one day, referred to as "1 min 3 times") exhibited a substantial and continuous re-release of Ca and P ions for 14 days without further recharge. The ion re-release did not decrease with increasing the number of recharge/re-release cycles (P>0.1). The ion re-release concentrations at 14 days versus various recharge treatments were as follows: 1 min 3 times>3 min 2 times>1 min 2 times>6 min 1 time>3 min 1 time>1 min 1 time. In conclusion, although previous studies have shown that NACP nanocomposite remineralized tooth lesions and inhibited caries, the present study developed the first orthodontic cement with Ca and P ion recharge and long-term release capability. This NACP-rechargeable orthodontic cement is a promising therapy to inhibit enamel demineralization and WSLs around orthodontic brackets.

Modelling cell cycle synchronisation in networks of coupled radial glial cells.

PubMed

Barrack, Duncan S; Thul, Rüdiger; Owen, Markus R

2015-07-21

Radial glial cells play a crucial role in the embryonic mammalian brain. Their proliferation is thought to be controlled, in part, by ATP mediated calcium signals. It has been hypothesised that these signals act to locally synchronise cell cycles, so that clusters of cells proliferate together, shedding daughter cells in uniform sheets. In this paper we investigate this cell cycle synchronisation by taking an ordinary differential equation model that couples the dynamics of intracellular calcium and the cell cycle and extend it to populations of cells coupled via extracellular ATP signals. Through bifurcation analysis we show that although ATP mediated calcium release can lead to cell cycle synchronisation, a number of other asynchronous oscillatory solutions including torus solutions dominate the parameter space and cell cycle synchronisation is far from guaranteed. Despite this, numerical results indicate that the transient and not the asymptotic behaviour of the system is important in accounting for cell cycle synchronisation. In particular, quiescent cells can be entrained on to the cell cycle via ATP mediated calcium signals initiated by a driving cell and crucially will cycle in near synchrony with the driving cell for the duration of neurogenesis. This behaviour is highly sensitive to the timing of ATP release, with release at the G1/S phase transition of the cell cycle far more likely to lead to near synchrony than release during mid G1 phase. This result, which suggests that ATP release timing is critical to radial glia cell cycle synchronisation, may help us to understand normal and pathological brain development. Copyright © 2015 Elsevier Ltd. All rights reserved.

A flow-system comparison of the reactivities of calcium superoxide and potassium superoxide with carbon dioxide and water vapor

NASA Technical Reports Server (NTRS)

Wood, P. C.; Ballou, E. V.; Spitze, L. A.; Wydeven, T.

1982-01-01

A single pass flow system was used to test the reactivity of calcium superoxide with respiratory gases and the performance was compared to that of potassium superoxide. The KO2 system is used by coal miners as a self-contained unit in rescue operations. Particular attention was given to the reactivity with carbon dioxide and water vapor at different temperatures and partial pressures of oxygen, carbon dioxide, and water vapor. The calcium superoxide beds were found to absorb CO2 and H2O vapor, releasing O2. The KO2 bed, however, released O2 at twice the rate of CO2 absorption at 37 C. It is concluded that the calcium superoxide material is not a suitable replacement for the KO2 bed, although Ca(O2)2 may be added to the KO2 bed to enhance the CO2 absorption.

DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

PubMed

Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

2014-04-01

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

Protein encapsulation and release from PEO-b-polyphosphoester templated calcium carbonate particles.

PubMed

Ergul Yilmaz, Zeynep; Cordonnier, Thomas; Debuigne, Antoine; Calvignac, Brice; Jerome, Christine; Boury, Frank

2016-11-20

Calcium carbonate particles are promising candidates as proteins carriers for their controlled delivery in the body. The present paper aims at investigating the protein encapsulation by in situ precipitation of calcium carbonate particles prepared by a process based on supercritical CO 2 and using a new type of degradable well-defined double hydrophilic block copolymers composed of poly(ethylene oxide) and polyphosphoester blocks acting as templating agent for the calcium carbonate. For this study, lysozyme was chosen as a model for therapeutic protein for its availability and ease of detection. It was found that by this green process, loading into the CaCO 3 microparticles with a diameter about 2μm can be obtained as determined by scanning electron microscopy. A protein loading up to 6.5% active lysozyme was measured by a specific bioassay (Micrococcus lysodeikticus). By encapsulating fluorescent-labelled lysozyme (lysozyme-FITC), the confocal microscopy images confirmed its encapsulation and suggested a core-shell distribution of lysozyme into CaCO 3 , leading to a release profile reaching a steady state at 59% of release after 90min. Copyright © 2016. Published by Elsevier B.V.

(Z)3,4,5,4‧-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level

NASA Astrophysics Data System (ADS)

Fan, Xing-Xing; Yao, Xiao-Jun; Xu, Su Wei; Wong, Vincent Kam-Wai; He, Jian-Xing; Ding, Jian; Xue, Wei-Wei; Mujtaba, Tahira; Michelangeli, Francesco; Huang, Min; Huang, Jun; Xiao, Da-Kai; Jiang, Ze-Bo; Zhou, Yan-Ling; Kin-Ting Kam, Richard; Liu, Liang; Lai-Han Leung, Elaine

2015-11-01

Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4‧-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients.

A ROS-Assisted Calcium Wave Dependent on the AtRBOHD NADPH Oxidase and TPC1 Cation Channel Propagates the Systemic Response to Salt Stress.

PubMed

Evans, Matthew J; Choi, Won-Gyu; Gilroy, Simon; Morris, Richard J

2016-07-01

Plants exhibit rapid, systemic signaling systems that allow them to coordinate physiological and developmental responses throughout the plant body, even to highly localized and quickly changing environmental stresses. The propagation of these signals is thought to include processes ranging from electrical and hydraulic networks to waves of reactive oxygen species (ROS) and cytoplasmic Ca(2+) traveling throughout the plant. For the Ca(2+) wave system, the involvement of the vacuolar ion channel TWO PORE CHANNEL1 (TPC1) has been reported. However, the precise role of this channel and the mechanism of cell-to-cell propagation of the wave have remained largely undefined. Here, we use the fire-diffuse-fire model to analyze the behavior of a Ca(2+) wave originating from Ca(2+) release involving the TPC1 channel in Arabidopsis (Arabidopsis thaliana). We conclude that a Ca(2+) diffusion-dominated calcium-induced calcium-release mechanism is insufficient to explain the observed wave transmission speeds. The addition of a ROS-triggered element, however, is able to quantitatively reproduce the observed transmission characteristics. The treatment of roots with the ROS scavenger ascorbate and the NADPH oxidase inhibitor diphenyliodonium and analysis of Ca(2+) wave propagation in the Arabidopsis respiratory burst oxidase homolog D (AtrbohD) knockout background all led to reductions in Ca(2+) wave transmission speeds consistent with this model. Furthermore, imaging of extracellular ROS production revealed a systemic spread of ROS release that is dependent on both AtRBOHD and TPC1 These results suggest that, in the root, plant systemic signaling is supported by a ROS-assisted calcium-induced calcium-release mechanism intimately involving ROS production by AtRBOHD and Ca(2+) release dependent on the vacuolar channel TPC1. © 2016 American Society of Plant Biologists. All Rights Reserved.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

PubMed Central

Wu, Zhenyong; Thiyagarajan, Sathish; O’Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible. PMID:29066949

The Different Facets of Extracellular Calcium Sensors: Old and New Concepts in Calcium-Sensing Receptor Signalling and Pharmacology

PubMed Central

2018-01-01

The current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca2+ changes. Widely recognized as a fundamental player in systemic Ca2+ homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+ microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+ sensors is provided. PMID:29584660

6-OHDA induced calcium influx through N-type calcium channel alters membrane properties via PKA pathway in substantia nigra pars compacta dopaminergic neurons.

PubMed

Qu, Liang; Wang, Yuan; Zhang, Hai-Tao; Li, Nan; Wang, Qiang; Yang, Qian; Gao, Guo-Dong; Wang, Xue-Lian

2014-07-11

Voltage gated calcium channels (VGCC) are sensitive to oxidative stress, and their activation or inactivation can impact cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remain controversial. In this study, we assessed 6-hydroxydopamine (6-OHDA) induced transformation of firing pattern and functional changes of calcium channels in SNc dopaminergic neurons. Application of 6-OHDA (0.5-2mM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)]i) rise. In voltage clamp, ω-conotoxin-sensitive Ca(2+) current modulation mediated by 6-OHDA reflected an altered sensitivity. Furthermore, we found that 6-OHDA modulated Ca(2+) currents through PKA pathway. These results provided evidence for the potential role of VGCCs and PKA involved in oxidative stress in degeneration of SNc neurons in Parkinson's disease (PD). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

PubMed Central

Liao, Yajin; Dong, Yuan; Cheng, Jinbo

2017-01-01

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. PMID:28208618

Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Binod; Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085; Kumar, Amit

2012-01-13

Highlights: Black-Right-Pointing-Pointer Diospyrin diethylether (D7) caused oxidative stress-dependent activation of PC-PLC. Black-Right-Pointing-Pointer Activated PC-PLC induced a sustained-release of Ca{sup 2+} from endoplasmic reticulum. Black-Right-Pointing-Pointer The elevated cytosolic Ca{sup +2} led to the calpain-caspase12 dependent apoptosis. Black-Right-Pointing-Pointer D7-Induced Ca{sup +2} also found to accentuate the mitochondrial pathway of apoptosis. -- Abstract: Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca{sup 2+}]{sub c} leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells.more » A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca{sup 2+}]{sub c} and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca{sup 2+} mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca{sup 2+}]{sub c} which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca{sup 2+}-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca{sup 2+}]{sub c} was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca{sup 2+} uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.« less

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

PubMed Central

Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis. PMID:25917787

Lattice model for calcium dynamics

NASA Astrophysics Data System (ADS)

Guisoni, Nara; de Oliveira, Mario José

2005-06-01

We present a simplified lattice model to study calcium dynamics in the endoplasmic reticulum membrane. Calcium channels and calcium ions are placed in two interpenetrating square lattices which are connected in two ways: (i) via calcium release and (ii) because transitions between channel states are calcium dependent. The opening or closing of a channel is a stochastic process controlled by two functions which depend on the calcium density on the channel neighborhood. The model is studied through mean field calculations and simulations. We show that the critical behavior of the model changes drastically depending on the opening/closing functions. For certain choices of these functions, all channels are closed at very low and high calcium densities and the model presents one absorbing state.

Alteration of Motor Network Function Following Injury

DTIC Science & Technology

2012-10-01

mechanisms from neuromodulator- dependent corre- lations of mRNA and conductance levels. Ion channel conductances are correlated across channel types...de- pendent on intracellular calcium signaling mechanisms that depend on the release of intracellular calcium stores. Because relatively rapid changes...changes in K current mag- nitudes are independently regulated by distinct mechanisms . Compensatory increases in IKCa are calcium- dependent and due, at

No association between the vitamin D pathway gene polymorphisms and bone biomarkers response to calcium and low dose calcitriol supplementation in postmenopausal Chinese women: a one-year prospective study.

PubMed

Gu, Jiemei; Wang, Chun; Zhang, Hao; Yue, Hua; Hu, Weiwei; He, Jinwei; Fu, Wenzhen; Zhang, Zhenlin

2018-05-18

The aim of the study was to explore the association between the vitamin D pathway gene variations and the bone biomarkers response to calcium and low dose calcitriol supplementation in postmenopausal Chinese women. A total of 110 healthy postmenopausal Chinese women (61.51 ± 6.93 years) were enrolled. The participants were supplemented with calcium (600 mg/d) and calcitriol (0.25 μg/d), for 1 year. Four biomarkers, serum levels of beta C-terminal cross-linked telopeptides of type I collagen (β-CTX), amino-terminal propeptide of type I collagen (P1NP), parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] were measured at baseline and 12-month follow-up. Multivariate regression models were established to explore the statistical association between the change rate of the four biomarkers and 15 key genes within the vitamin D metabolic pathway. This exclusion process left 98 participants for analysis. Serum levels of P1NP, β-CTX and PTH were significantly decreased at the 12-month follow-up (all p < 0.05). Serum 25(OH)D level had no significant change (p > 0.05). No association was found between the vitamin D pathway gene polymorphisms and bone biomarkers response to calcium and low dose calcitriol supplementation. Genetic background of postmenopausal Chinese women might not influence supplemental response of the biomarkers to calcium and low dose calcitriol.

Calcium movements during pigment aggregation in freshwater shrimp chromatophores.

PubMed

Ribeiro, Márcia; McNamara, John Campbell

2007-02-01

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

PubMed Central

Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

2015-01-01

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP3 receptors (IP3Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP3R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP3R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP3R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment. PMID:25637353

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

PubMed

Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

2015-03-12

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment. © 2015 Institut Curie/Inserm. Published under the terms of the CC BY NC ND 4.0 license.

Regulation of Spinal Substance P Release by Intrathecal Calcium Channel Blockade

PubMed Central

Takasusuki, Toshifumi; Yaksh, Tony L.

2012-01-01

Background We investigated the role of different voltage sensitive calcium channels expressed at presynaptic afferent terminals in substance P release and on nociceptive behavior evoked by intraplantar formalin by examining the effects of intrathecally delivered N- (ziconotide), T- (mibefradil) and L-type voltage sensitive calcium channels blockers (diltiazem and verapamil). Methods Rats received intrathecal pretreatment with saline or doses of morphine, ziconotide, mibefradil, diltiazem or verapamil. The effect of these injections upon flinching evoked by intraplantar formalin (5%, 50μl) was quantified. To assess substance P release, the incidence of neurokinin 1 receptor internalization in the ipsilateral and contralateral lamina I was determined in immunofluorescent stained tissues. Results Intrathecal morphine (20μg), ziconotide (0.3, 0.6 and 1μg), mibefradil (100μg, but not 50μg), diltiazem (500μg, but not 300μg) and verapamil (200μg, but not 50 and 100μg) reduced paw flinching in phase 2 as compared to vehicle control (P < 0.05), with no effect upon phase 1. Ziconotide (0.3, 0.6 and 1μg) and morphine (20μg) significantly inhibited neurokinin 1 receptor internalization (P < 0.05), but mibefradil, diltiazem and verapamil at the highest doses had no effect. Conclusion These results emphasize the role in vivo of N-, but not T- and L-type voltage sensitive calcium channels in mediating the stimulus evoked substance P release from small primary afferents and suggest that T- and L-type voltage sensitive calcium channels blockers exert antihyperalgesic effects by an action on other populations of afferents or mechanisms involving post synaptic excitability. PMID:21577088

Calexcitin interaction with neuronal ryanodine receptors.

PubMed Central

Nelson, T J; Zhao, W Q; Yuan, S; Favit, A; Pozzo-Miller, L; Alkon, D L

1999-01-01

Calexcitin (CE), a Ca2+- and GTP-binding protein, which is phosphorylated during memory consolidation, is shown here to co-purify with ryanodine receptors (RyRs) and bind to RyRs in a calcium-dependent manner. Nanomolar concentrations of CE released up to 46% of the 45Ca label from microsomes preloaded with 45CaCl2. This release was Ca2+-dependent and was blocked by antibodies against the RyR or CE, by the RyR inhibitor dantrolene, and by a seven-amino-acid peptide fragment corresponding to positions 4689-4697 of the RyR, but not by heparin, an Ins(1,4,5)P3-receptor antagonist. Anti-CE antibodies, in the absence of added CE, also blocked Ca2+ release elicited by ryanodine, suggesting that the CE and ryanodine binding sites were in relative proximity. Calcium imaging with bis-fura-2 after loading CE into hippocampal CA1 pyramidal cells in hippocampal slices revealed slow, local calcium transients independent of membrane depolarization. Calexcitin also released Ca2+ from liposomes into which purified RyR had been incorporated, indicating that CE binding can be a proximate cause of Ca2+ release. These results indicated that CE bound to RyRs and suggest that CE may be an endogenous modulator of the neuronal RyR. PMID:10393102

Retention and release of oil-in-water emulsions from filled hydrogel beads composed of calcium alginate: impact of emulsifier type and pH.

PubMed

Zeeb, Benjamin; Saberi, Amir Hossein; Weiss, Jochen; McClements, David Julian

2015-03-21

Delivery systems based on filled hydrogel particles (microgels) can be fabricated from natural food-grade lipids and biopolymers. The potential for controlling release characteristics by modulating the electrostatic interactions between emulsifier-coated lipid droplets and the biopolymer matrix within hydrogel particles was investigated. A multistage procedure was used to fabricate calcium alginate beads filled with lipid droplets stabilized by non-ionic, cationic, anionic, or zwitterionic emulsifiers. Oil-in-water emulsions stabilized by Tween 60, DTAB, SDS, or whey protein were prepared by microfluidization, mixed with various alginate solutions, and then microgels were formed by simple extrusion into calcium solutions. The microgels were placed into a series of buffer solutions with different pH values (2 to 11). Lipid droplets remained encapsulated under acidic and neutral conditions, but were released under highly basic conditions (pH 11) due to hydrogel swelling when the alginate concentration was sufficiently high. Lipid droplet release increased with decreasing alginate concentration, which could be attributed to an increase in the pore size of the hydrogel matrix. These results have important implications for the design of delivery systems to entrap and control the release of lipophilic bioactive components within filled hydrogel particles.

High glucose-induced Ca2+ overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways.

PubMed

Kumar, Sandeep; Kain, Vasundhara; Sitasawad, Sandhya L

2012-07-01

Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting. H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis. High-glucose treatment resulted in increased intracellular calcium ([Ca2+]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca2+]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G0/G1 DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death. This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy. The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium. Copyright © 2012 Elsevier B.V. All rights reserved.

Herpes simplex virus type 2 glycoprotein H interacts with integrin αvβ3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

PubMed

Cheshenko, Natalia; Trepanier, Janie B; González, Pablo A; Eugenin, Eliseo A; Jacobs, William R; Herold, Betsy C

2014-09-01

Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The in vivo anti-fibrotic function of calcium sensitive receptor (CaSR) modulating poly(p-dioxanone-co-l-phenylalanine) prodrug.

PubMed

Wang, Bing; Wen, Aiping; Feng, Chengmin; Niu, Lijing; Xiao, Xin; Luo, Le; Shen, Chengyi; Zhu, Jiang; Lei, Jun; Zhang, Xiaoming

2018-06-01

In present study, the apoptosis induction and proliferation suppression effects of l-phenylalanine (l-Phe) on fibroblasts were confirmed. The action sites of l-Phe on fibroblasts suppression were deduced to be calcium sensitive receptor (CaSR) which could cause the release of endoplasmic reticulum (ER) Ca 2+ stores; disruption of intracellular Ca 2+ homeostasis triggers cell apoptosis via the ER or mitochondrial pathways. The down-regulation of CaSR were observed after the application of l-Phe, and the results those l-Phe triggered the increasing of intracellular Ca 2+ concentration and calcineurin expression, and then the apoptosis and increasing G1 fraction of fibroblasts have verified our deduction. Hence, l-Phe could be seen as a kind of anti-fibrotic drugs for the crucial participation of fibroblast in the occurrence of fibrosis. And then, poly(p-dioxanone-co-l-phenylalanine) (PDPA) which could prolong the in-vivo anti-fibrotic effect of l-Phe for the sustained release of l-Phe during its degradation could be treated as anti-fibrotic polymer prodrugs. Based on the above, the in vivo anti-fibrotic function of PDPA was evaluated in rabbit ear scarring, rat peritoneum lipopolysaccharide, and rat sidewall defect/cecum abrasion models. PDPA reduced skin scarring and suppressed peritoneal fibrosis and post operation adhesion as well as secretion of transforming growth factor-β1 in injured tissue. These results indicate that PDPA is an effective agent for preventing fibrosis following tissue injury. We have previously demonstrated that poly(p-dioxanone-co-l-phenylalanine) (PDPA) could induce apoptosis to fibroblast and deduced that the inhibitory effect comes from l-phenylalanine. In present study, the inhibition mechanism of l-phenylalanine on fibroblast proliferation was demonstrated. The calcium sensitive receptor (CaSR) was found to be the action site. The CaSR was downregulated after the application of l-phenylalanine, and then the ER Ca 2+ stores were released. The released Ca 2+ can simultaneously activate Ca 2+ /calcineurin and then trigger apoptosis and G1 arrest of fibroblast. Hence, l-phenylalanine could be seen as anti-fibrosis drug and PDPA which conjugate l-phenylalanine by hydrolytic covalent bonds could be seen as l-phenylalanine polymer prodrug. Based above, the in vivo anti-fibrotic function of PDPA were verified in three different animal models. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Endotrophic Calcium, Strontium, and Barium Spores of Bacillus megaterium and Bacillus cereus1

PubMed Central

Foerster, Harold F.; Foster, J. W.

1966-01-01

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333–1345. 1966.—Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl2, SrCl2, or BaCl2. Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed “coat fraction” from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH. Images PMID:4956334

Plant innate immunity – sunny side up?

PubMed Central

Stael, Simon; Kmiecik, Przemyslaw; Willems, Patrick; Van Der Kelen, Katrien; Coll, Nuria S.; Teige, Markus; Van Breusegem, Frank

2016-01-01

Reactive oxygen species (ROS)- and calcium- dependent signaling pathways play well-established roles during plant innate immunity. Chloroplasts host major biosynthetic pathways and have central roles in energy production, redox homeostasis, and retrograde signaling. However, the organelle’s importance in immunity has been somehow overlooked. Recent findings suggest that the chloroplast also has an unanticipated function as a hub for ROS- and calcium-signaling that affects immunity responses at an early stage after pathogen attack. In this opinion article, we discuss a chloroplastic calcium-ROS signaling branch of plant innate immunity. We propose that this chloroplastic branch acts as a light-dependent rheostat that, through the production of ROS, influences the severity of the immune response. PMID:25457110

Diagram of Calcium Movement in the Human Body

NASA Technical Reports Server (NTRS)

2002-01-01

This diagram shows the normal pathways of calcium movement in the body and indicates changes (green arrows) seen during preliminary space flight experiments. Calcium plays a central role because 1) it gives strength and structure to bone and 2) all types of cells require it to function normally. To better understand how and why weightlessness induces bone loss, astronauts have participated in a study of calcium kinetics -- that is, the movement of calcium through the body, including absorption from food, and its role in the formation and breakdown of bone.

Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Derian, C.K.; Friedman, P.A.

1988-04-01

The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in (Ca2+)i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release /sup 45/Ca from intracellular sites in permeabilized cells. We showmore » here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced /sup 45/Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.« less

Hypoxia-mimicking bioactive glass regenerative effects on dental stem cells

NASA Astrophysics Data System (ADS)

Noor, Siti Noor Fazliah Mohd; Azevedo, Maria; Mohamad, Hasmaliza; Autefage, Hélène

2016-12-01

Vascularization is an important aspect of tissue regeneration. Hypoxia, low oxygen concentration, is a known stimulus for the release of vascular endothelial growth factors (VEGF) which play important roles in vascularization. The current study aimed to assess the effect of a cobalt-containing bioactive glass (BG) in stimulating hypoxia and promoting vascularization. To incorporate cobalt into BG, 1 mol% of calcium was substituting with cobalt, and this formulation was compared to the one without cobalt. Both BGs were processed via melt-derived method. The BG powders with particle size less than 38 µm were incubated with cell culture medium for 4 hours at 37°C on continuous rolling, and then the medium was filtered using 0.22 µm syringe filters. Prior to use, the BG-conditioned media were supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic-antimycotic, and were allowed to equilibrate overnight inside a CO2 incubator. The conditioned media were used on human dental stem cells (stem cells from permanent (DPSC) and deciduous (SHED) teeth) and assessed for their capacity to stimulate the release of angiogenic factors from the cells. The results showed that cobalt ions were released from the cobalt-containing BG, following partial dissolution of the glasses in cell culture medium, and promoted VEGF release from the cells. In conclusion, the incorporation of cobalt in BG may have potential to be used for tissue regeneration by promoting vascularization through the activation of hypoxia pathway and the release of VEGF.

Facilitation of granule cell epileptiform activity by mossy fiber-released zinc in the pilocarpine model of temporal lobe epilepsy.

PubMed

Timofeeva, Olga; Nadler, J Victor

2006-03-17

Recurrent mossy fiber synapses in the dentate gyrus of epileptic brain facilitate the synchronous firing of granule cells and may promote seizure propagation. Mossy fiber terminals contain and release zinc. Released zinc inhibits the activation of NMDA receptors and may therefore oppose the development of granule cell epileptiform activity. Hippocampal slices from rats that had experienced pilocarpine-induced status epilepticus and developed a recurrent mossy fiber pathway were used to investigate this possibility. Actions of released zinc were inferred from the effects of chelation with 1 mM calcium disodium EDTA (CaEDTA). When granule cell population bursts were evoked by mossy fiber stimulation in the presence of 6 mM K(+) and 30 microM bicuculline, CaEDTA slowed the rate at which evoked bursting developed, but did not change the magnitude of the bursts once they had developed fully. The effects of CaEDTA were then studied on the pharmacologically isolated NMDA receptor- and AMPA/kainate receptor-mediated components of the fully developed bursts. CaEDTA increased the magnitude of NMDA receptor-mediated bursts and reduced the magnitude of AMPA/kainate receptor-mediated bursts. CaEDTA did not affect the granule cell bursts evoked in slices from untreated rats by stimulating the perforant path in the presence of bicuculline and 6 mM K(+). These results suggest that zinc released from the recurrent mossy fibers serves mainly to facilitate the recruitment of dentate granule cells into population bursts.

Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of beta-cell muscarinic receptor activation on cAMP production.

PubMed

Dolz, Manuel; Bailbé, Danielle; Giroix, Marie-Hélène; Calderari, Sophie; Gangnerau, Marie-Noelle; Serradas, Patricia; Rickenbach, Katharina; Irminger, Jean-Claude; Portha, Bernard

2005-11-01

Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK beta-cell and, in doing so, normalizes its defective glucose responsiveness.

Enhanced drug encapsulation and extended release profiles of calcium-alginate nanoparticles by using tannic acid as a bridging cross-linking agent.

PubMed

Abulateefeh, Samer R; Taha, Mutasem O

2015-01-01

Calcium alginate nanoparticles (NPs) suffer from sub-optimal stability in bio-relevant media leading to low drug encapsulation efficiency and uncontrolled release profiles. To sort out these drawbacks, a novel approach is proposed herein based on introducing tannic acid into these NPs to act as a bridging cross-linking aid agent. Calcium-alginate NPs were prepared by the ionotropic gelation method and loaded with diltiazem hydrochloride as a model drug. These NPs were characterized in terms of particle size, zeta potential, and morphology, and results were explained in accordance with Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The incorporation of tannic acid led to more than four folds increase in drug encapsulation efficiency (i.e. from 15.3% to 69.5%) and reduced burst drug release from 44% to around 10% within the first 30 min. These findings suggest the possibility of improving the properties of Ca-alginate NPs by incorporating cross-linking aid agents under mild conditions.

Blends of jackfruit seed starch-pectin in the development of mucoadhesive beads containing metformin HCl.

PubMed

Nayak, Amit Kumar; Pal, Dilipkumar

2013-11-01

In this work, calcium pectinate-jackfruit (Artocarpus heterophyllus Lam.) seed starch (JFSS) mucoadhesive beads containing metformin HCl were developed through ionotropic-gelation. Effects of pectin and JFSS amounts on drug encapsulation efficiency (DEE), and cumulative drug release after 10 h (R10 h) were optimized using 3(2) factorial design. The optimized calcium pectinate-JFSS beads containing metformin HCl showed DEE of 94.11 ± 3.92%, R10 h of 48.88 ± 2.02%, and mean diameter of 2.06 ± 0.20 mm. The in vitro drug release from these beads was followed controlled-release (zero-order) pattern with super case-II transport mechanism. The beads were also characterized by SEM and FTIR. The pH of test mediums was found critical for swelling and mucoadhesion of these beads. The optimized calcium pectinate-JFSS beads also exhibited good mucoadhesivity and significant hypoglycemic effect in alloxan-induced diabetic rats over prolonged period after oral administration. Copyright © 2013 Elsevier B.V. All rights reserved.

Calcium addition at the Hubbard Brook Experimental Forest increases sugar storage, antioxidant activity and cold tolerance in native red spruce (Picea rubens)

Treesearch

Joshua M. Halman; Paul G. Schaberg; Gary J. Hawley; Christopher Eagar

2008-01-01

In fall (November 2005) and winter (February 2006), we collected current-year foliage of native red spruce (Picea rubens Sarg.) growing in a reference watershed and in a watershed treated in 1999 with wollastonite (CaSiO3, a slow-release calcium source) to simulate preindustrial soil calcium concentrations (Ca-addition...

Development of the foremost light-curable calcium-silicate MTA cement as root-end in oral surgery. Chemical-physical properties, bioactivity and biological behavior.

PubMed

Gandolfi, Maria Giovanna; Taddei, Paola; Siboni, Francesco; Modena, Enrico; Ciapetti, Gabriela; Prati, Carlo

2011-07-01

An innovative light-curable calcium-silicate cement containing a HEMA-TEGDMA-based resin (lc-MTA) was designed to obtain a bioactive fast setting root-end filling and root repair material. lc-MTA was tested for setting time, solubility, water absorption, calcium release, alkalinizing activity (pH of soaking water), bioactivity (apatite-forming ability) and cell growth-proliferation. The apatite-forming ability was investigated by micro-Raman, ATR-FTIR and ESEM/EDX after immersion at 37°C for 1-28 days in DPBS or DMEM+FBS. The marginal adaptation of cement in root-end cavities of extracted teeth was assessed by ESEM/EDX, and the viability of Saos-2 cell on cements was evaluated. lc-MTA demonstrated a rapid setting time (2min), low solubility, high calcium release (150-200ppm) and alkalinizing power (pH 10-12). lc-MTA proved the formation of bone-like apatite spherulites just after 1 day. Apatite precipitates completely filled the interface porosities and created a perfect marginal adaptation. lc-MTA allowed Saos-2 cell viability and growth and no compromising toxicity was exerted. HEMA-TEGDMA creates a polymeric network able to stabilize the outer surface of the cement and a hydrophilic matrix permeable enough to allow water absorption. SiO(-)/Si-OH groups from the mineral particles induce heterogeneous nucleation of apatite by sorption of calcium and phosphate ions. Oxygen-containing groups from poly-HEMA-TEGDMA provide additional apatite nucleating sites through the formation of calcium chelates. The strong novelty was that the combination of a hydraulic calcium-silicate powder and a poly-HEMA-TEGDMA hydrophilic resin creates the conditions (calcium release and functional groups able to chelate Ca ions) for a bioactive fast setting light-curable material for clinical applications in dental and maxillofacial surgery. The first and unique/exclusive light-curable calcium-silicate MTA cement for endodontics and root-end application was created, with a potential strong impact on surgical procedures. Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle.

PubMed

Fusi, L; Brunello, E; Yan, Z; Irving, M

2016-10-31

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway.

Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle

PubMed Central

Fusi, L.; Brunello, E.; Yan, Z.; Irving, M.

2016-01-01

Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin filaments is controlled by the stress in the myosin-containing thick filaments. Those results suggested that thick filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of thick filament activation depend on thick filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by thick filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway. PMID:27796302

Influence of Chemical, Mechanical, and Transport Processes on Wellbore Leakage from Geologic CO2 Storage Reservoirs.

PubMed

Carroll, Susan A; Iyer, Jaisree; Walsh, Stuart D C

2017-08-15

Wells are considered to be high-risk pathways for fluid leakage from geologic CO 2 storage reservoirs, because breaches in this engineered system have the potential to connect the reservoir to groundwater resources and the atmosphere. Given these concerns, a few studies have assessed leakage risk by evaluating regulatory records, often self-reported, documenting leakage in gas fields. Leakage is thought to be governed largely by initial well-construction quality and the method of well abandonment. The geologic carbon storage community has raised further concerns because acidic fluids in the CO 2 storage reservoir, alkaline cement meant to isolate the reservoir fluids from the overlying strata, and steel casings in wells are inherently reactive systems. This is of particular concern for storage of CO 2 in depleted oil and gas reservoirs with numerous legacy wells engineered to variable standards. Research suggests that leakage risks are not as great as initially perceived because chemical and mechanical alteration of cement has the capacity to seal damaged zones. Our work centers on defining the coupled chemical and mechanical processes governing flow in damaged zones in wells. We have developed process-based models, constrained by experiments, to better understand and forecast leakage risk. Leakage pathways can be sealed by precipitation of carbonate minerals in the fractures and deformation of the reacted cement. High reactivity of cement hydroxides releases excess calcium that can precipitate as carbonate solids in the fracture network under low brine flow rates. If the flow is fast, then the brine remains undersaturated with respect to the solubility of calcium carbonate minerals, and zones depleted in calcium hydroxides, enriched in calcium carbonate precipitates, and made of amorphous silicates leached of original cement minerals are formed. Under confining pressure, the reacted cement is compressed, which reduces permeability and lowers leakage risks. The broader context of this paper is to use our experimentally calibrated chemical, mechanical, and transport model to illustrate when, where, and in what conditions fracture pathways seal in CO 2 storage wells, to reduce their risk to groundwater resources. We do this by defining the amount of cement and the time required to effectively seal the leakage pathways associated with peak and postinjection overpressures, within the context of oil and gas industry standards for leak detection, mitigation, and repairs. Our simulations suggest that for many damage scenarios chemical and mechanical processes lower leakage risk by reducing or sealing fracture pathways. Leakage risk would remain high in wells with a large amount of damage, modeled here as wide fracture apertures, where fast flowing fluids are too dilute for carbonate precipitation and subsurface stress does not compress the altered cement. Fracture sealing is more likely as reservoir pressures decrease during the postinjection phase where lower fluxes aid chemical alteration and mechanical deformation of cement. Our results hold promise for the development of mitigation framework to avoid impacting groundwater resources above any geologic CO 2 storage reservoir by correlating operational pressures and barrier lengths.

Influence of Chemical, Mechanical, and Transport Processes on Wellbore Leakage from Geologic CO 2 Storage Reservoirs

DOE PAGES

Carroll, Susan A.; Iyer, Jaisree; Walsh, Stuart D. C.

2017-07-25

Wells are considered to be high-risk pathways for fluid leakage from geologic CO 2 storage reservoirs, because breaches in this engineered system have the potential to connect the reservoir to groundwater resources and the atmosphere. Given these concerns, a few studies have assessed leakage risk by evaluating regulatory records, often self-reported, documenting leakage in gas fields. Leakage is thought to be governed largely by initial well-construction quality and the method of well abandonment. The geologic carbon storage community has raised further concerns because acidic fluids in the CO 2 storage reservoir, alkaline cement meant to isolate the reservoir fluids frommore » the overlying strata, and steel casings in wells are inherently reactive systems. This is of particular concern for storage of CO 2 in depleted oil and gas reservoirs with numerous legacy wells engineered to variable standards. Research suggests that leakage risks are not as great as initially perceived because chemical and mechanical alteration of cement has the capacity to seal damaged zones. Our work centers on defining the coupled chemical and mechanical processes governing flow in damaged zones in wells. We have developed process-based models, constrained by experiments, to better understand and forecast leakage risk. Leakage pathways can be sealed by precipitation of carbonate minerals in the fractures and deformation of the reacted cement. High reactivity of cement hydroxides releases excess calcium that can precipitate as carbonate solids in the fracture network under low brine flow rates. If the flow is fast, then the brine remains undersaturated with respect to the solubility of calcium carbonate minerals, and zones depleted in calcium hydroxides, enriched in calcium carbonate precipitates, and made of amorphous silicates leached of original cement minerals are formed. Under confining pressure, the reacted cement is compressed, which reduces permeability and lowers leakage risks. The broader context of this paper is to use our experimentally calibrated chemical, mechanical, and transport model to illustrate when, where, and in what conditions fracture pathways seal in CO 2 storage wells, to reduce their risk to groundwater resources. We do this by defining the amount of cement and the time required to effectively seal the leakage pathways associated with peak and postinjection overpressures, within the context of oil and gas industry standards for leak detection, mitigation, and repairs. Our simulations suggest that for many damage scenarios chemical and mechanical processes lower leakage risk by reducing or sealing fracture pathways. Leakage risk would remain high in wells with a large amount of damage, modeled here as wide fracture apertures, where fast flowing fluids are too dilute for carbonate precipitation and subsurface stress does not compress the altered cement. Fracture sealing is more likely as reservoir pressures decrease during the postinjection phase where lower fluxes aid chemical alteration and mechanical deformation of cement. Our results hold promise for the development of mitigation framework to avoid impacting groundwater resources above any geologic CO 2 storage reservoir by correlating operational pressures and barrier lengths.« less

Influence of Chemical, Mechanical, and Transport Processes on Wellbore Leakage from Geologic CO 2 Storage Reservoirs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carroll, Susan A.; Iyer, Jaisree; Walsh, Stuart D. C.

Wells are considered to be high-risk pathways for fluid leakage from geologic CO 2 storage reservoirs, because breaches in this engineered system have the potential to connect the reservoir to groundwater resources and the atmosphere. Given these concerns, a few studies have assessed leakage risk by evaluating regulatory records, often self-reported, documenting leakage in gas fields. Leakage is thought to be governed largely by initial well-construction quality and the method of well abandonment. The geologic carbon storage community has raised further concerns because acidic fluids in the CO 2 storage reservoir, alkaline cement meant to isolate the reservoir fluids frommore » the overlying strata, and steel casings in wells are inherently reactive systems. This is of particular concern for storage of CO 2 in depleted oil and gas reservoirs with numerous legacy wells engineered to variable standards. Research suggests that leakage risks are not as great as initially perceived because chemical and mechanical alteration of cement has the capacity to seal damaged zones. Our work centers on defining the coupled chemical and mechanical processes governing flow in damaged zones in wells. We have developed process-based models, constrained by experiments, to better understand and forecast leakage risk. Leakage pathways can be sealed by precipitation of carbonate minerals in the fractures and deformation of the reacted cement. High reactivity of cement hydroxides releases excess calcium that can precipitate as carbonate solids in the fracture network under low brine flow rates. If the flow is fast, then the brine remains undersaturated with respect to the solubility of calcium carbonate minerals, and zones depleted in calcium hydroxides, enriched in calcium carbonate precipitates, and made of amorphous silicates leached of original cement minerals are formed. Under confining pressure, the reacted cement is compressed, which reduces permeability and lowers leakage risks. The broader context of this paper is to use our experimentally calibrated chemical, mechanical, and transport model to illustrate when, where, and in what conditions fracture pathways seal in CO 2 storage wells, to reduce their risk to groundwater resources. We do this by defining the amount of cement and the time required to effectively seal the leakage pathways associated with peak and postinjection overpressures, within the context of oil and gas industry standards for leak detection, mitigation, and repairs. Our simulations suggest that for many damage scenarios chemical and mechanical processes lower leakage risk by reducing or sealing fracture pathways. Leakage risk would remain high in wells with a large amount of damage, modeled here as wide fracture apertures, where fast flowing fluids are too dilute for carbonate precipitation and subsurface stress does not compress the altered cement. Fracture sealing is more likely as reservoir pressures decrease during the postinjection phase where lower fluxes aid chemical alteration and mechanical deformation of cement. Our results hold promise for the development of mitigation framework to avoid impacting groundwater resources above any geologic CO 2 storage reservoir by correlating operational pressures and barrier lengths.« less

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Serotonin and the regulation of calcium transport in dairy cows.

PubMed

Hernandez, L L

2017-12-01

The mammary gland regulates maternal metabolism during lactation. Numerous factors within the tissue send signals to shift nutrients to the mammary gland for milk synthesis. Serotonin is a monoamine that has been well documented to regulate several aspects of lactation among species. Maintenance of maternal calcium homeostasis during lactation is a highly evolved process that is elegantly regulated by the interaction of the mammary gland with the bone, gut, and kidney tissues. It is well documented that dietary calcium is insufficient to maintain maternal calcium concentrations during lactation, and mammals must rely on bone resorption to maintain normocalcemia. Our recent work focused on the ability of the mammary gland to function as an accessory parathyroid gland during lactation. It was demonstrated that serotonin acts to stimulate parathyroid hormone-related protein (PTHrP) in the mammary gland during lactation. The main role of mammary-derived PTHrP during mammalian lactation is to stimulate bone resorption to maintain maternal calcium homeostasis during lactation. In addition to regulating PTHrP, it was shown that serotonin appears to directly affect calcium transporters and pumps in the mammary gland. Our current working hypothesis regarding the control of calcium during lactation is as follows: serotonin directly stimulates PTHrP production in the mammary gland through interaction with the sonic hedgehog signaling pathway. Simultaneously, serotonin directly increases calcium movement into the mammary gland and, subsequently, milk. These 2 direct actions of serotonin combine to induce a transient maternal hypocalcemia required to further stimulate PTHrP production and calcium mobilization from bone. Through these 2 routes, serotonin is able to improve maternal calcium concentrations. Furthermore, we have shown that Holstein and Jersey cows appear to regulate calcium in different manners and also respond differently to serotonergic stimulation of the calcium pathway. Our data in rodents and cows indicate that serotonin and calcium are working through a unique feedback loop with PTHrP during lactation to regulate milk calcium and maternal calcium homeostasis.

Cell-type-specific modelling of intracellular calcium signalling: a urothelial cell model.

PubMed

Appleby, Peter A; Shabir, Saqib; Southgate, Jennifer; Walker, Dawn

2013-09-06

Calcium signalling plays a central role in regulating a wide variety of cell processes. A number of calcium signalling models exist in the literature that are capable of reproducing a variety of experimentally observed calcium transients. These models have been used to examine in more detail the mechanisms underlying calcium transients, but very rarely has a model been directly linked to a particular cell type and experimentally verified. It is important to show that this can be achieved within the general theoretical framework adopted by these models. Here, we develop a framework designed specifically for modelling cytosolic calcium transients in urothelial cells. Where possible, we draw upon existing calcium signalling models, integrating descriptions of components known to be important in this cell type from a number of studies in the literature. We then add descriptions of several additional pathways that play a specific role in urothelial cell signalling, including an explicit ionic influx term and an active pumping mechanism that drives the cytosolic calcium concentration to a target equilibrium. The resulting one-pool model of endoplasmic reticulum (ER)-dependent calcium signalling relates the cytosolic, extracellular and ER calcium concentrations and can generate a wide range of calcium transients, including spikes, bursts, oscillations and sustained elevations in the cytosolic calcium concentration. Using single-variate robustness and multivariate sensitivity analyses, we quantify how varying each of the parameters of the model leads to changes in key features of the calcium transient, such as initial peak amplitude and the frequency of bursting or spiking, and in the transitions between bursting- and plateau-dominated modes. We also show that, novel to our urothelial cell model, the ionic and purinergic P2Y pathways make distinct contributions to the calcium transient. We then validate the model using human bladder epithelial cells grown in monolayer cell culture and show that the model robustly captures the key features of the experimental data in a way that is not possible using more generic calcium models from the literature.

Calcium signaling in taste cells: regulation required.

PubMed

Medler, Kathryn F

2010-11-01

Peripheral taste receptor cells depend on distinct calcium signals to generate appropriate cellular responses that relay taste information to the central nervous system. Some taste cells have conventional chemical synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release from stores to formulate an output signal through a hemichannel. Despite the importance of calcium signaling in taste cells, little is known about how these signals are regulated. This review summarizes recent studies that have identified 2 calcium clearance mechanisms expressed in taste cells, including mitochondrial calcium uptake and sodium/calcium exchangers (NCXs). These studies identified a unique constitutive calcium influx that contributes to maintaining appropriate calcium homeostasis in taste cells and the role of the mitochondria and exchangers in this process. The additional role of NCXs in the regulation of evoked calcium responses is also discussed. Clearly, calcium signaling is a dynamic process in taste cells and appears to be more complex than has previously been appreciated.

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

PubMed

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.

Transcriptome Analysis of Calcium- and Hormone-Related Gene Expressions during Different Stages of Peanut Pod Development

PubMed Central

Li, Yan; Meng, Jingjing; Yang, Sha; Guo, Feng; Zhang, Jialei; Geng, Yun; Cui, Li; Wan, Shubo; Li, Xinguo

2017-01-01

Peanut is one of the calciphilous plants. Calcium serves as a ubiquitous central hub in a large number of signaling pathways. In the field, free calcium ion (Ca2+)-deficient soil can result in unfilled pods. Four pod stages were analyzed to determine the relationship between Ca2+ excretion and pod development. Peanut shells showed Ca2+ excretion at all four stages; however, both the embryo of Stage 4 (S4) and the red skin of Stage 3 (S3) showed Ca2+ absorbance. These results showed that embryo and red skin of peanut need Ca2+ during development. In order to survey the relationship among calcium, hormone and seed development from gene perspective, we further analyzed the seed transcriptome at Stage 2 (S2), S3, and S4. About 70 million high quality clean reads were generated, which were assembled into 58,147 unigenes. By comparing these three stages, total 4,457 differentially expressed genes were identified. In these genes, 53 Ca2+ related genes, 40 auxin related genes, 15 gibberellin genes, 20 ethylene related genes, 2 abscisic acid related genes, and 7 cytokinin related genes were identified. Additionally, a part of them were validated by qRT-PCR. Most of their expressions changed during the pod development. Since some reports showed that Ca2+ signal transduction pathway is involved in hormone regulation pathway, these results implied that peanut seed development might be regulated by the collaboration of Ca2+ signal transduction pathway and hormone regulation pathway. PMID:28769950

Effects of copyrolysis of sludge with calcium carbonate and calcium hydrogen phosphate on chemical stability of carbon and release of toxic elements in the resultant biochars.

PubMed

Xu, Xuebin; Hu, Xin; Ding, Zhuhong; Chen, Yijun

2017-12-01

The potential release of toxic elements and the stability of carbon in sludge-based biochars are important on their application in soil remediation and wastewater treatment. In this study, municipal sludge was co-pyrolyzed with calcium carbonate (CaCO 3 ) and calcium dihydrogen phosphate [Ca(H 2 PO 4 ) 2 ] under 300 and 600 °C, respectively. The basic physicochemical properties of the resultant biochars were characterized and laboratory chemical oxidation and leaching experiments of toxic elements were conducted to evaluate the chemical stability of carbon in biochars and the potential release of toxic elements from biochars. Results show that the exogenous minerals changed the physico-chemical properties of the resultant biochars greatly. Biochars with exogenous minerals, especially Ca(H 2 PO 4 ) 2 , decreased the release of Zn, Cr, Ni, Cu, Pb, and As and the release ratios were less than 1%. Tessier's sequential extraction analysis revealed that labile toxic elements were transferred to residual fraction in the biochars with high pyrolysis temperature (600 °C) and exogenous minerals. Low risks for biochar-bound Pb, Zn, Cd, As, Cr, and Cu were confirmed according to risk assessment code (RAC) while the potential ecological risk index (PERI) revealed that the exogenous Ca(H 2 PO 4 ) 2 significantly decreased the risks from considerable to moderate level. Moreover, the exogenous minerals significantly increased the chemical stability of carbon in 600 °C-pyrolyzed biochars by 10-20%. These results indicated that the copyrolysis of sludge with phosphate and carbonate, especially phosphate, were effective methods to prepare the sludge-based biochars with immobilized toxic elements and enhanced chemical stability of carbon. Copyright © 2017 Elsevier Ltd. All rights reserved.

AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis

PubMed Central

Hsu, Ron-Bin; Shun, Chia-Tung; Hsu, Chih-Chieh

2017-01-01

ABSTRACT Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis. PMID:28674029

Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

PubMed

Danese, Alberto; Patergnani, Simone; Bonora, Massimo; Wieckowski, Mariusz R; Previati, Maurizio; Giorgi, Carlotta; Pinton, Paolo

2017-08-01

Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca 2+ ) release from the ER allows selective Ca 2+ uptake by the mitochondria. The perturbation of Ca 2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca 2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

Mucin 4 Gene Silencing Reduces Oxidative Stress and Calcium Oxalate Crystal Formation in Renal Tubular Epithelial Cells Through the Extracellular Signal-Regulated Kinase Signaling Pathway in Nephrolithiasis Rat Model.

PubMed

Sun, Ling; Zou, Lu-Xi; Wang, Jie; Chen, Ting; Han, Yu-Chen; Zhu, Dong-Dong; Zhuo, Shi-Chao

2018-05-25

Nephrolithiasis plagues a great number of patients all over the world. Increasing evidence shows that the extracellular signal-regulated kinase (ERK) signaling pathway and renal tubular epithelial cell (RTEC) dysfunction and attrition are central to the pathogenesis of kidney diseases. Mucin 4 (MUC4) is reported as an activator of ERK signaling pathway in epithelial cells. In this study, using rat models of calcium oxalate (CaOx) nephrolithiasis, the present study aims to define the roles of MUC4 and ERK signaling pathway as contributors to oxidative stress and CaOx crystal formation in RTEC. Data sets of nephrolithiasis were searched using GEO database and a heat flow map was drawn. Then MUC4 function was predicted. Wistar rats were prepared for the purpose of model establishment of ethylene glycol and ammonium chloride induced CaOx nephrolithiasis. In order to assess the detailed regulatory mechanism of MUC4 silencing on the ERK signaling pathway and RTEC, we used recombinant plasmid to downregulate MUC4 expression in Wistar rat-based models. Samples from rat urine, serum and kidney tissues were reviewed to identify oxalic acid and calcium contents, BUN, Cr, Ca2+ and P3+ levels, calcium crystal formation in renal tubules and MUC4 positive expression rate. Finally, RT-qPCR, Western blot analysis, and ELISA were employed to access oxidative stress state and CaOx crystal formation in RTEC. Initially, MUC4 was found to have an influence on the process of nephrolithiasis. MUC4 was upregulated in the CaOx nephrolithiasis model rats. We proved that the silencing of MUC4 triggered the inactivation of ERK signaling pathway. Following the silencing of MUC4 or the inhibition of ERK signaling pathway, the oxalic acid and calcium contents in rat urine, BUN, Cr, Ca2+ and P3+ levels in rat serum, p-ERK1/2, MCP-1 and OPN expressions in RTEC and H2O2 and MDA levels in the cultured supernatant were downregulated, but the GSH-Px, CAT and SOD levels in the cultured supernatant were increased. Moreover, MUC4 silencing or ERK signaling pathway inactivation may decrease the formation of CaOx crystals. Taken together, silencing of MUC4 can inactivate the ERK signaling pathway and further restrain oxidative stress and CaOx crystal formation in RTEC. Thus, MUC4 represents a potential investigative focus target in nephrolithiasis. © 2018 The Author(s). Published by S. Karger AG, Basel.

The Effect of Formulation Excipients and Thermal Treatment on the Release Properties of Lisinopril Spheres and Tablets.

PubMed

Amador Ríos, Zoriely; Ghaly, Evone Shehata

2015-01-01

Multiparticulate systems are used in the development of controlled release systems. The objective of this study was to determine the effect of the wax level, the type of excipient, and the exposure of the tablets to thermal treatment on drug release. Spheres from multiparticulate system with different wax levels and excipients were developed using the drug Lisinopril and compressed into tablets; these tablets were analyzed to determine the drug release. All tablets contained constant level of Lisinopril (10% w/w) and Compritol (30% and 50% w/w). Also, as a diluent, all of them contained 30% w/w Avicel and 30% w/w dibasic calcium phosphate or lactose, or 60% Avicel. Tablets compacted from spheres prepared by extruder/marumerizer and using 30% w/w lipid and 60% Avicel released 84% of drug at six hours of dissolution testing, while tablets of the same composition but prepared using 30% dibasic calcium phosphate and 30% Avicel released 101%. When the tablets were thermally treated, the drug release reduced. As the percent of lipid increased in the formulation, the drug release decreased. Compaction of tablets prepared from spheres with wax has potential for controlling the drug release.

Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury

PubMed Central

Thamilselvan, Vijayalakshmi; Menon, Mani

2013-01-01

Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence. PMID:21814770

Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

PubMed Central

Wen, Xianjie; Xu, Shiyuan; Liu, Hongzhen; Zhang, Quinguo; Liang, Hua; Yang, Chenxiang; Wang, Hanbing

2013-01-01

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation. PMID:23658789

Caffeine alleviates the deterioration of Ca2+ release mechanisms and fragmentation of in vitro aged mouse eggs

PubMed Central

Zhang, Nan; Wakai, Takuya; Fissore, Rafael. A.

2011-01-01

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others found that in these eggs the intracellular calcium ([Ca2+]i) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca2+ release is not well known. Here, we investigated if the function of IP3R1, the major Ca2+ release channel at fertilization, was undermined in in vitro aged mouse eggs. We found that in aged eggs IP3R1 displayed reduced function, as many of the changes acquired during maturation that enhance IP3R1 Ca2+ conductivity such as phosphorylation, receptor reorganization and increased Ca2+ store content ([Ca2+]ER) were lost with increasing postovulatory time. IP3R1 fragmentation, possibly associated with the activation of caspase-3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP3R1 function and maintained [Ca2+]ER content. Caffeine also maintained mitochondrial membrane potential as measured by JC-1 fluorescence. We therefore conclude that [Ca2+]i responses in aged eggs are undermined by reduced IP3R1 sensitivity, decreased [Ca2+]ER and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging-associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs. PMID:22095868

Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling

PubMed Central

Dobson, Katharine L.; Jackson, Claire; Balakrishnan, Saju; Bellamy, Tomas C.

2015-01-01

Background Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites—a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. Methods Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. Key Results Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine—intracellular calcium release, and cAMP signalling—had no impact on these effects. Conclusions We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections. PMID:25933382

Phase Composition Control of Calcium Phosphate Nanoparticles for Tunable Drug Delivery Kinetics and Treatment of Osteomyelitis. Part 1: Preparation and Drug Release

PubMed Central

Uskoković, Vuk; Desai, Tejal A.

2012-01-01

Developed in this study is a multifunctional material for simultaneous osseoinduction and drug delivery, potentially applicable in the treatment of osteomyelitis. It is composed of agglomerates of nanoparticles of calcium phosphate (CAP) with different monophasic contents. The drug loading capacity and the release kinetics were investigated on two model drug compounds with different chemical structures, sizes and adsorption propensities: bovine serum albumin and fluorescein. Loading of CAP powders with small molecule drugs was achieved by physisorption and desiccation-induced agglomeration of nanoparticulate subunits into microscopic blocks. The material dissolution rate and the drug release rate depended on the nature of the CAP phase, decreasing from monocalcium phosphate to monetite to amorphous CAP and calcium pyrophosphate to hydroxyapatite. The sustained release of the two model drugs was shown to be directly relatable to the degradation rate of CAP carriers. It was demonstrated that the degradation rate of the carrier and the drug release kinetics could be made tunable within the time scale of 1–2 h for the most soluble CAP phase, monocalcium phosphate, to 1–2 years for the least soluble one, hydroxyapatite. From the standpoint of antibiotic therapy for osteomyelitis, typically lasting for six weeks, the most prospective CAP powder was amorphous CAP with its release time scale for a small organic molecule, the same category to which antibiotics belong, of 1 – 2 months under the conditions applied in our experiments. By combining these different CAP phases in various proportions, drug release profiles could be tailored to the therapeutic occasion. PMID:23115118

LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

PubMed

Kwon, Seok-Kyu; Sando, Richard; Lewis, Tommy L; Hirabayashi, Yusuke; Maximov, Anton; Polleux, Franck

2016-07-01

Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization.

PubMed

Matroule, J Y; Carthy, C M; Granville, D J; Jolois, O; Hunt, D W; Piette, J

2001-07-05

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

Altering the level of calcium changes the physical properties and digestibility of casein-based emulsion gels.

PubMed

McIntyre, Irene; O Sullivan, Michael; O Riordan, Dolores

2017-04-19

Casein-based emulsion gels prepared with different types of lipid (i.e. milk fat or rapeseed oil) were formulated with high (774 mg Ca per 100 g) or low (357 mg Ca per 100 g) calcium levels by blending acid and rennet casein. Their physicochemical characteristics (i.e. composition, texture, microstructure & water mobility) and in vitro digestibility were compared to conventionally formulated high-calcium (723 mg Ca per 100 g) emulsion gels made from rennet casein with calcium chelating salts (CCS). CCS-free, high-calcium emulsion gels were significantly (p ≤ 0.05) softer than those with low calcium levels (possibly due to their shorter manufacture time and higher pH) and showed the highest rates of disintegration during simulated gastric digestion. Despite having a higher moisture to protein ratio, the high-calcium emulsion gels containing CCS had broadly similar hardness values to those of high-calcium concentration prepared without CCS, but had higher cohesiveness. The high-calcium matrices containing CCS had quite a different microstructure and increased water mobility compared to those made without CCS and showed the slowest rate (p ≤ 0.05) of disintegration in the gastric environment. Gastric resistance was not affected by the type of lipid phase. Conversely, fatty acid release was similar for all emulsion gels prepared from milk fat, however, high-calcium emulsion gels (CCS-free) prepared from rapeseed oil showed higher lipolysis. Results suggest that food matrix physical properties can be modified to alter resistance to gastric degradation which may have consequences for the kinetics of nutrient release and delivery of bioactives sensitive to the gastric environment.

Ion release, fluoride charge of and adhesion of an orthodontic cement paste containing microcapsules.

PubMed

Burbank, Brant D; Slater, Michael; Kava, Alyssa; Doyle, James; McHale, William A; Latta, Mark A; Gross, Stephen M

2016-02-01

Dental materials capable of releasing calcium, phosphate and fluoride are of great interest for remineralization. Microencapsulated aqueous solutions of these ions in orthodontic cement demonstrate slow, sustained release by passive diffusion through a permeable membrane without the need for dissolution or etching of fillers. The potential to charge a dental material formulated with microencapsulated water with fluoride by toothbrushing with over the counter toothpaste and the effect of microcapsules on cement adhesion to enamel was determined. Orthodontic cements that contained microcapsules with water and controls without microcapsules were brushed with over-the-counter toothpaste and fluoride release was measured. Adhesion measurements were performed loading orthodontic brackets to failure. Cements that contained microencapsulated solutions of 5.0M Ca(NO3)2, 0.8M NaF, 6.0MK2HPO4 or a mixture of all three were prepared. Ion release profiles were measured as a function of time. A greater fluoride charge and re-release from toothbrushing was demonstrated compared to a control with no microcapsules. Adhesion of an orthodontic cement that contained microencapsulated remineralizing agents was 8.5±2.5MPa compared to the control without microcapsules which was of 8.3±1.7MPa. Sustained release of fluoride, calcium and phosphate ions from cement formulated with microencapsulated remineralizing agents was demonstrated. Orthodontic cements with microcapsules show a release of bioavailable fluoride, calcium, and phosphate ions near the tooth surface while having the ability to charge with fluoride and not effect the adhesion of the material to enamel. Incorporation of microcapsules in dental materials is promising for promoting remineralization. Copyright © 2015 Elsevier Ltd. All rights reserved.

Signal transduction networks and the biology of plant cells.

PubMed

Chrispeels, M J; Holuigue, L; Latorre, R; Luan, S; Orellana, A; Peña-Cortes, H; Raikhel, N V; Ronald, P C; Trewavas, A

1999-01-01

The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Biotechnology

NASA Image and Video Library

2002-07-31

This diagram shows the normal pathways of calcium movement in the body and indicates changes (green arrows) seen during preliminary space flight experiments. Calcium plays a central role because 1) it gives strength and structure to bone and 2) all types of cells require it to function normally. To better understand how and why weightlessness induces bone loss, astronauts have participated in a study of calcium kinetics -- that is, the movement of calcium through the body, including absorption from food, and its role in the formation and breakdown of bone.

Prostaglandin D2 regulates human colonic ion transport via the DP1 receptor.

PubMed

Medani, M; Collins, D; Mohan, H M; Walsh, E; Winter, D C; Baird, A W

2015-02-01

Prostaglandin D2 is released by mast cells and is important in allergies. Its role in gastrointestinal function is not clearly defined. This study aimed to determine the effect of exogenous PGD2 on ion transport in ex vivo normal human colonic mucosa. Mucosal sheets were mounted in Ussing chambers and voltage clamped to zero electric potential. Ion transport was quantified as changes in short-circuit current. In separate experiments epithelial monolayers or colonic crypts, isolated by calcium chelation, were treated with PGD2 and cAMP levels determined by ELISA or calcium levels were determined by fluorimetry. PGD2 caused a sustained, concentration-dependent rise in short-circuit current by increasing chloride secretion (EC50=376nM). This effect of PGD2 is mediated by the DP1 receptor, as the selective DP1 receptor antagonist BW A686C inhibited PGD2-induced but not PGE2-induced rise in short-circuit current. PGD2 also increased intracellular cAMP in isolated colonic crypts with no measurable influence on cytosolic calcium. PGD2 induces chloride secretion in isolated human colonic mucosa in a concentration-dependent manner with concomitant elevation of cytoplasmic cAMP in epithelial cells. The involvement of DP2 receptor subtypes has not previously been considered in regulation of ion transport in human intestine. Since inflammatory stimuli may induce production of eicosanoids, selective regulation of these pathways may be pivotal in determining therapeutic strategies and in understanding disease. Copyright © 2014. Published by Elsevier Inc.

Calcium transient in presynaptic terminal of squid giant synapse: detection with aequorin.

PubMed

Llinás, R; Blinks, J R; Nicholson, C

1972-06-09

Microinjection of aequorin, a bioluminescent protein sensitive tocalcium, into the presynaptic terminal of the squid giant synapse demnonstrated an increase in intracellular calcium ion concentration during repetitive synaptic transmission. Although no light flashes synchronous with individual presynaptic : tion potentials were detected, the results are considered consistent with the hypothesis that entry of calcium into the presynaptic terminal triggers release of e synaptic transmitter substance.

Interactions between Electromagnetic Fields.

DTIC Science & Technology

1985-02-10

the cortical surface of a cat, in vivo, caused calcium release. Follow-up.nvestigatlons, using radiofrequency (RF) radiation , utilized simplfer, in...vitro brain tissue preparations from the cat and from the chicken. RF radiation was found to cause changes in the calcium flux only when the radiation ...circumvented by the finding th#’ t sub-ELF signals applied directly to the sample can also cause changes ip the calcium fluxes, although at tissue intensities on

Glutamate Signaling and Mitochondrial Dysfunction in Models of Parkinson’s Disease

DTIC Science & Technology

2014-03-01

stages of PD, an elevation in synaptically released glutamate leads to persistent activation of NMDARs that synergizes with Cav1 calcium channels to...neurons is attributable to activity -dependent calcium entry through Cav1 channels, resulting in mitochondrial oxidant stress. Although this mechanism...glutamate leads to persistent activation of NMDARs that synergizes with Cav1 calcium channels to significantly increase mitochondrial oxidant stress and

Understanding spatial and temporal patterning of astrocyte calcium transients via interactions between network transport and extracellular diffusion

NASA Astrophysics Data System (ADS)

Shtrahman, E.; Maruyama, D.; Olariu, E.; Fink, C. G.; Zochowski, M.

2017-02-01

Astrocytes form interconnected networks in the brain and communicate via calcium signaling. We investigate how modes of coupling between astrocytes influence the spatio-temporal patterns of calcium signaling within astrocyte networks and specifically how these network interactions promote coordination within this group of cells. To investigate these complex phenomena, we study reduced cultured networks of astrocytes and neurons. We image the spatial temporal patterns of astrocyte calcium activity and quantify how perturbing the coupling between astrocytes influences astrocyte activity patterns. To gain insight into the pattern formation observed in these cultured networks, we compare the experimentally observed calcium activity patterns to the patterns produced by a reduced computational model, where we represent astrocytes as simple units that integrate input through two mechanisms: gap junction coupling (network transport) and chemical release (extracellular diffusion). We examine the activity patterns in the simulated astrocyte network and their dependence upon these two coupling mechanisms. We find that gap junctions and extracellular chemical release interact in astrocyte networks to modulate the spatiotemporal patterns of their calcium dynamics. We show agreement between the computational and experimental findings, which suggests that the complex global patterns can be understood as a result of simple local coupling mechanisms.

Effects of adrenalectomy on the control and adrenergic regulation of cytosolic free calcium in hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freudenrich, C.C.

1987-01-01

The purpose of this study was to investigate the effects of adrenalectomy on the control and ..cap alpha..-adrenergic regulation of the concentration of cytosolic free calcium (Ca/sub i/) in hepatocytes. In hepatocytes isolated from adrenalectomized (adx) and sham-operated male rats 7-1 days after surgery, Ca/sub i/ at rest and in response to epinephrine (EPI) was measured with the calcium-sensitive photoprotein aequorin, /sup 45/Ca efflux was measured, and Ca/sup 2 +/ release from intracellular stores in response to inositol triphosphate (IP/sub 3/) was measured in saponin-permeabilized cells. Liver calmodulin content was also assayed by radioimmunoassay. It was found in adx ratsmore » that the resting Ca/sub i/ was elevated, the rise in Ca/sub i/ during EPI stimulation was reduced at physiological EPI concentrations, and the rise in calcium efflux evoked by EPI was reduced. Furthermore, the slope of the relationship between Ca/sub i/ and calcium efflux was reduced 60% in adx. Adx did not alter the characteristics of Ca/sup 2 +/ release from intracellular calcium pools in response to IP/sub 3/ in permeabilized cells. Finally, the liver calmodulin contents were not significantly different between the 2 groups.« less

Activation of apoptotic pathway in normal, cancer ovarian cells by epothilone B.

PubMed

Rogalska, Aneta; Szula, Ewa; Gajek, Arkadiusz; Marczak, Agnieszka; Jóźwiak, Zofia

2013-09-01

The epothilones, a new class of microtubule-targeting agents, seem to be a very promising alternative to the current strategy of cancer treatment. We have analyzed the aspects of epothilone B (Epo B) on cellular metabolism of tumor (OV-90) and normal (MM 14) ovarian cells. The observed effects were compared with those of paclitaxel (PTX), which is now a standard for the treatment of ovarian cancer. The results provide direct evidence that Epo B is considerably more cytotoxic to human OV-90 ovarian cancer cells than PTX. We have found, that antitumor efficacy of this new drug is related to its apoptosis-inducing ability, which was confirmed during measurements typical markers of the process. Epo B induced changes in morphology of cells, mitochondrial membrane potential and cytochrome c release. Also a slight increase of the intracellular calcium level was observed. Moreover, we have found that ROS production, stimulated by Epo B, is directly involved in the induction of apoptosis via mitochondrial pathway. Copyright © 2013 Elsevier B.V. All rights reserved.