Free Calcium Release Research Articles

In vitro digestion of solid-in-oil-in-water emulsions for delivery of CaCO3

The formation of CaCO3 loaded particles based on solid/oil/water (S/O/W) emulsions is a promising method to improve the dispersion stability and digestive properties of CaCO3 in liquid foods. Used CaCO3 as solid phase, soybean oil as O phase, sodium caseinate (NaCas), gelatin (GEL), and sodium caseate-gelatin (NaCas-GEL) as W phase, S/O/W calcium-lipid microspheres were prepared. The intermolecular interactions were examined by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and X-ray diffraction (XRD) analysis. GIT model was used to analyze the particle size, Zeta-potential, and microstructure at different digestion stages by combining the fatty acid release rate, the degree of protein hydrolysis, and the release of free calcium. The FTIR and Raman and XRD results showed that NaCas interacts with GEL to a certain extent, which was helpful for the preparation of stable S/O/W emulsion and could effectively improve the embedding rate of CaCO3. GIT studies showed the addition of GEL weakened the looseness and openness of the aggregation structure in the gastric digestion stage, reduced the FFA release rate to a certain extent, and had a certain inhibitory effect on the digestion of soybean oil in the emulsion. The results showed that the digestion of S/O/W emulsion was the fastest when the W phase was NaCas, and the addition of GEL could significantly reduce the digestion.

The multifaceted role of SOCE in central synapses

Store-operated calcium entry (SOCE) is a mechanism for calcium influx through the plasma membrane in response to release of free calcium from the endoplasmic reticulum. Two recent studies revealed how SOCE regulates the exocytosis of neurotransmitter vesicles at central synapses

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice.

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wildtype Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wildtype ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT); inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high affinity receptor); and ITPR1-mediated cytoplasmic free calcium release in vitro. Additionally, we show that gene-transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

Car8 dorsal root ganglion expression and genetic regulation of analgesic responses are associated with a cis-eQTL in mice.

Carbonic anhydrase-8 (Car8 mouse gene symbol) is devoid of enzymatic activity, but instead functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) to regulate this intracellular calcium release channel important in synaptic functions and neuronal excitability. Causative mutations in ITPR1 and carbonic anhydrase-8 in mice and humans are associated with certain subtypes of spinal cerebellar ataxia (SCA). SCA mice are genetically deficient in dorsal root ganglia (DRG) Car8 expression and display mechanical and thermal hypersensitivity and susceptibility to subacute and chronic inflammatory pain behaviors. In this report, we show that DRG Car8 expression is variable across 25 naïve-inbred strains of mice, and this cis-regulated eQTL (association between rs27660559, rs27706398, and rs27688767 and DRG Car8 expression; P < 1 × 10-11) is correlated with nociceptive responses in mice. Next, we hypothesized that increasing DRG Car8 gene expression would inhibit intracellular calcium release required for morphine antinociception and might correlate with antinociceptive sensitivity of morphine and perhaps other analgesic agents. We show that mean DRG Car8 gene expression is directly related to the dose of morphine or clonidine needed to provide a half-maximal analgesic response (r = 0.93, P < 0.00002; r = 0.83, P < 0.0008, respectively), suggesting that greater DRG Car8 expression increases analgesic requirements. Finally, we show that morphine induces intracellular free calcium release using Fura 2 calcium imaging in a dose-dependent manner; V5-Car8 WT overexpression in NBL cells inhibits morphine-induced calcium increase. These findings highlight the 'morphine paradox' whereby morphine provides antinociception by increasing intracellular free calcium, while Car8 and other antinociceptive agents work by decreasing intracellular free calcium. This is the first study demonstrating that biologic variability associated with this cis-eQTL may contribute to differing analgesic responses through altered regulation of ITPR1-dependent calcium release in mice.

Platelet Reactivity in Hemophilia a. Flow Cytometry Study to Detect the Hemorragic Phenotype

INTRODUCTIONHemophilia A (HA) is a rare inherited genetic disorder linked to the X chromosome, characterized by joint bleeding in patients with severe disease with <1% of factor VIII (FVIII)(White et al. Thromb Haemost 2001). However, there are cases of severe HA with less bleeding than usual and it has been postulated that there must be other contributors to the hemorrhagic phenotype, including a platelet dysfunction (Van Bladel et al. Haematologica 2011).In this study we analyzed platelet reactivity in a cohort of 16 male patients with severe HA in prophylactic treatment by flow cytometry. The control group was 15 healthy male subjects.METHODSA longitudinal prospective observational study cohort was conducted. The study was performed according to the Declaration of Helsinki of 1975 (revised 1983), and was approved by the Ethics Committee of our Institution. Sixteen HA male patients without FVIII inhibitor aged 9-39 years (mean 22,75 years, SD 11,428) and 15 adult male healthy volunteers aged 26-48 years (mean 36,4 years, SD 6,6) were included. All subjects had normal platelet counts (150-450x103/µl). The blood was collected by antecubital venipuncture and avoiding the use of tourniquet to minimize the spontaneous activation of platelets during extraction. For HA patients blood samples were taken immediately before FVIII infusion (basal sample, 72h without FVIII) and 15 minutes after the infusion of FVIII. Levels of factor FVIII in plasma were determined using a chromogenic method. We analyzed platelet responsiveness, using the thrombin receptor activator peptide-6 (TRAP-6) as agonist, measuring the expression of CD62P and CD63 by flow cytometry. For microparticles analysis, percentages and number of events positive for CD62P and CD41 were gated for Annexin-V+ subpopulation. Endothelial cell-derived microparticles were identified as CD144+CD41-. To calculate the absolute count we considered the final volumes of each tube and it was expressed in events/µL for each subpopulation (Annexin-V +, CD41+, CD144+ and CD62P+). For the analysis of cytoplasmic free calcium release, whole blood was labelled with PerCP-Cy5.5-human anti-CD61 and the calcium sensitive dye Fluo-4-AM for 15 min at room temperature. Following adjustment of the basal fluorescence, samples were stimulated with the agonists TRAP-6 or adenine-5«-diphosphate sodium salt (ADP) and analyzed for 5 min by flow cytometer. Ionophore A23187 was used as a positive control for calcium mobilization. Afterwards mean fluorescence intensity values were analysed with the Kaluza flow cytometry analysis software (Beckman Coulter).RESULTSStatistical analysis was carried out using SPSS statistical software version 19.0.All values were expressed as mean ± SD for each experimental group.To compare the results in HA patients before and after administration of FVIII we used the t-Student test for means related, to compare HA patients and the control group we used the t-student test for independent samples. Data of calcium were analyzed by one-way analysis of variance followed by Tukey-Kramer for multiple comparisons. The minimum acceptable level of significance wasp<0.05.Basal levels of FVIII in plasma were4,58 ± 6,4%.The mean fluorescence intensity for CD62P after stimulation with TRAP was significantly lower in HA patients after infusion of FVIII compared to basal levels (p<0,05)(Table 1). Regarding microparticles (MPs), we detected higher expression of CD41+ MPs in HA patients after infusion of FVIII compared to basal expression, but not for CD41+CD62P+ MPs. Also, percentage of CD144+ MPs were significantly lower in HA patients after FVIII infusion (Table 2). Moreover, we did not find statistically significant differences comparing the release of calcium between the different groups (Table 3).CONCLUSIONIn this study severe HA patients in prophylactic treatment displayed normal platelet activation and microparticles plasmatic levels. However, the platelet activation marker CD62P after TRAP agonist and the endothelial cell origin microparticles expression were lower 15 minutes after the infusion factor VIII.AcknowledgmentsWe wish to thank to Pfizer providing economic support to this project. [Display omitted] [Display omitted] [Display omitted] DisclosuresMelero-Amor:Pfizer: Research Funding. García Candel:Pfizer: Research Funding. García:Pfizer: Research Funding. Romecin:Pfizer: Research Funding. Iyu:Pfizer: Research Funding. Cabañas-Perianes:Pfizer: Research Funding. Pérez:Pfizer: Research Funding. O´Connor:Pfizer: Research Funding. Moraleda:Pfizer: Research Funding. Marín:Pfizer: Research Funding. Blanquer Blanquer:Pfizer: Research Funding.

Carbonic anhydrase-8 regulates inflammatory pain by inhibiting the ITPR1-cytosolic free calcium pathway.

Calcium dysregulation is causally linked with various forms of neuropathology including seizure disorders, multiple sclerosis, Huntington’s disease, Alzheimer’s, spinal cerebellar ataxia (SCA) and chronic pain. Carbonic anhydrase-8 (Car8) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates intracellular calcium release fundamental to critical cellular functions including neuronal excitability, neurite outgrowth, neurotransmitter release, mitochondrial energy production and cell fate. In this report we test the hypothesis that Car8 regulation of ITPR1 and cytoplasmic free calcium release is critical to nociception and pain behaviors. We show Car8 null mutant mice (MT) exhibit mechanical allodynia and thermal hyperalgesia. Dorsal root ganglia (DRG) from MT also demonstrate increased steady-state ITPR1 phosphorylation (pITPR1) and cytoplasmic free calcium release. Overexpression of Car8 wildtype protein in MT nociceptors complements Car8 deficiency, down regulates pITPR1 and abolishes thermal and mechanical hypersensitivity. We also show that Car8 nociceptor overexpression alleviates chronic inflammatory pain. Finally, inflammation results in downregulation of DRG Car8 that is associated with increased pITPR1 expression relative to ITPR1, suggesting a possible mechanism of acute hypersensitivity. Our findings indicate Car8 regulates the ITPR1-cytosolic free calcium pathway that is critical to nociception, inflammatory pain and possibly other neuropathological states. Car8 and ITPR1 represent new therapeutic targets for chronic pain.

A Pivotal Role for the Multifunctional Calcium/Calmodulin-Dependent Protein Kinase II in T Cells: From Activation to Unresponsiveness

Stimulation of the TCR leads to an oscillatory release of free calcium that activates members of the calcium/calmodulin-dependent protein kinase II (CaMKII) family. The CaMKII molecules have profound and lasting effects on cellular signaling in several cell types, yet the role of CaMKII in T cells is still poorly characterized. In this report we describe a splice variant of CaMKIIbeta, CaMKIIbeta'e, in mouse T cells. We have determined its function, along with that of CaMKIIgamma, by introducing the active and kinase-dead mutants into activated P14 TCR transgenic T cells using retroviral transduction. Active CaMKII enhanced the proliferation and cytotoxic activity of T cells while reducing their IL-2 production. Furthermore, it induced a profound state of unresponsiveness that could be overcome only by prolonged culture in IL-2. These results indicate that members of the CaMKII family play an important role in regulation of CD8 T cell proliferation, cytotoxic effector function, and the response to restimulation.

A novel murine model of orthopaedic wear‐debris associated osteolysis

Objective: To develop a mouse model of bone resorption to quantitatively evaluate wear‐debris induced osteolysis.Methods: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of femur or calvaria from a syngeneic mouse donor. One group of bone‐implanted pouches was stimulated with ultra‐high molecular weight polyethylene (UHMWPE) debris, and the remaining bone‐implanted pouches received saline alone as controls. The tissues were harvested at 2, 7, and 14 days after bone implantation for molecular and histological analyses.Results: Marked inflammatory responses (thicker membrane and increased cellular infiltration) were observed in UHMWPE‐stimulated pouches, compared with the saline control. Intensive tartrate‐resistant acid phosphatase (TRAP) staining was identified in the UHMWPE‐stimulated pouches, especially at the attachment site of inflammatory tissue with implanted bone, where active osteolysis occurred. Image analysis showed that the bone collagen loss was closely related to the amount of UHMWPE within the tissue, and was most prevalent at the contact site of bone with inflammatory tissue. UHMWPE stimulation also significantly increased the release of free calcium into the pouch fluids.Conclusion: This model demonstrates a sensitive, rapid, and reproducible method for studying wear‐debris induced osteolysis seen in patients with aseptic loosening.

Nuclear versus perinuclear and cytoplasmic calcium in osteoclasts

We measured fluorescence from the calcium indicator Fluo-3 in multinucleated osteoclasts. In the initial state, each nucleus is surrounded by a ring of bright fluorescence. Following activation of purinergic receptors by 100 μM ATP there is a pulse of cellular fluorescence increase, and nuclear fluorescence intensity becomes greater than that of the cytoplasm. This is followed by a period during which the fluorescence of the cell decreases below that of the initial state. During the pulsed increase following purinergic receptor activation, the perinuclear fluorescence intensity does not increase as much as that in the nuclear centers and, following this pulse, the perinuclear fluorescence intensity decreases more than that in the nuclear centers, relative to the initial state. Measurements in which Mn 2+ was introduced into the cell show that the number of Fluo-3 molecules per unit horizontal area in the nuclear centers is slightly greater than that in the perinuclear regions, and more than twice that in the surrounding cytoplasm. These results show that there is a much higher free calcium concentration in the perinuclear regions than in the nuclear centers in the initial state, with a release of free calcium from the perinuclear regions following activation of the purinergic receptors. These data also provide evidence that the free calcium concentration in the nuclear centers is lower than in the cytoplasm in the initial state.

Leukotriene B4, an arachidonic acid metabolite, regulates intracellular free calcium release in eggs and mitotic cells of the sand dollar (Echinaracnius parma).

Previous articleNext article No AccessCELL DIVISION, CELL MOTILITY, AND DEVELOPMENTLeukotriene B4, an Arachidonic Acid Metabolite, Regulates Intracellular Free Calcium Release in Eggs and Mitotic Cells of the Sand Dollar (Echinaracnius parma)R. B. Silver, J. B. Oblak, G. S. Jeun, J. J. Sung, and T. C. DuttaR. B. Silver Search for more articles by this author , J. B. Oblak Search for more articles by this author , G. S. Jeun Search for more articles by this author , J. J. Sung Search for more articles by this author , and T. C. Dutta Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 187, Number 2October 1994 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv187n2p242 Views: 10Total views on this site Copyright © 1994 by Marine Biological LaboratoryPDF download Crossref reports the following articles citing this article:Robert A. Knudson, Shinya Inoué, and Makoto Goda Centrifuge Polarizing Microscope with Dual Specimen Chambers and Injection Ports, The Biological Bulletin 201, no.22 (Sep 2016): 234–234.https://doi.org/10.2307/1543339Robert B. Silver LtB4 Evokes the Calcium Signal That Initiates Nuclear Envelope Breakdown through a Multi-enzyme Network in Sand Dollar (Echinaracnius parma) Cells, The Biological Bulletin 201, no.22 (Sep 2016): 248–250.https://doi.org/10.2307/1543348R. B. Silver and N. M. Deming Leukotriene B4 as Calcium Agonist for Nuclear Envelope Breakdown: An Enzymological Survey of Endomembranes of Mitotic Cells, The Biological Bulletin 197, no.22 (Sep 2016): 268–270.https://doi.org/10.2307/1542644R. B. Silver, D. E. Strongin, L. R. Hurwitz, and A. P. Reeves Identification of Phospholipase A2 and Phospholipase C Activities in Calcium Regulatory Endomembranes of Sand Dollar Cells, The Biological Bulletin 193, no.22 (Sep 2016): 236–237.https://doi.org/10.1086/BBLv193n2p236 R. B. Silver Leukotriene B4 Induces Release of Calcium From Endomembrane Stores In Vivo in Eggs and Second Cell Cycle Blastomeres of the Sand Dollar Echinaracnius parma, The Biological Bulletin 189, no.22 (Sep 2016): 203–204.https://doi.org/10.1086/BBLv189n2p203 M. Krendel and S. Inoué Anaphase Spindle Dynamics Under D20-enhanced Microtubule Polymerization, The Biological Bulletin 189, no.22 (Sep 2016): 204–205.https://doi.org/10.1086/BBLv189n2p204

Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells.

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.

Multiple Sclerosis Brain Immunoglobulins Stimulate Myelin Basic Protein Degradation in Human Myelin: A New Cause of Demyelination

Membrane-bound proteolysis may be implicated in the pathogenesis of demyelinating disorders including multiple sclerosis (MS). We previously found that the extent of myelin basic protein (MBP) degradation by the calcium-activated neutral protease did not differ for isolated human control myelin or MS myelin. Hence we suggested that, if involved in demyelination, the myelin neutral protease must be activated in vivo by an increased availability of free calcium. The postulate was therefore tested that immunoglobulin (Ig) binding to myelin results in activation of the myelin neutral protease, possibly through release of free calcium from calcium-binding sites of myelin. Isolated myelin from the brains of controls and patients with MS were incubated with purified Igs eluted from the brains of patients with MS or controls and degradation of MBP was assessed by quantitative electroimmunoblotting. Such degradation was significantly greater in myelin incubated in the presence of MS Igs than in myelin incubated without added Igs or in the presence of control Igs. Furthermore, the degree of MBP degradation in myelin incubated with control Igs was similar to that observed in myelin incubated without added Igs. Accordingly, it is suggested that Ig in MS brain potentiates myelin breakdown. Moreover activation of membrane-bound proteolysis by Ig binding to myelin appears to represent a hitherto undescribed pathway for demyelination in MS.

Free calcium wave upon activation in Xenopus eggs

Eggs of Xenopus laevis were preloaded with aequorin and the spatial and temporal pattern of free calcium release in the egg cortex on artificial activation was determined by the aequorin luminescence emitted from the thin cortical layer of naturally opaque eggs. The aequorin luminescence was detected with a photonic microscope system consisting of a light microscope and a two-dimensional photon-counting system with an image processor. A free calcium increase was initiated around the point of prick activation. The state of increased Ca 2+ propagated in the cortical cytoplasm of the egg as a wave with a velocity of about 8 μm/sec at 22°C. This wave reached the antipode by 5 to 6 min of prick activation. The spatial pattern of the Ca 2+ wave was similar to that of changes in brightness of the egg surface on activation, termed the “activation wave” by K. Hara and P. Tydeman (1979, Wilhelm Roux's Arch. Dev. Biol., 186, 91–94). To examine the temporal correlation between the Ca 2+ wave and the activation wave, images of aequorin luminescence and those of the egg cortex taken by incident light illumination were recorded alternately in the same egg. The zone of free calcium increase corresponded to the light (relaxation) zone of the activation wave, where exocytosis of cortical granules and elongation of microvilli were taking place.

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs: (Ca2+ /fertilization/medaka/sand dollar/aequorin).

A transient rise in the concentration of Ca2+ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca2+ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2-3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca2+ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca2+ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca2+ into the cortex also induced a Ca2+ wave. When the egg was stimulated by microinjection of Ca2+ at the equatorial region, the Ca2+ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca2+ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca2+ wave preceded the wave of cortical change. A Ca2+ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.

Voltage transients elicited by sudden step‐up of auxin

It is hypothesized (i) that the molecular mechanism for the reception of friction and flexure and the mechanism by which auxin enhances ethylene production have in common a release of free calcium into the cytosol, (ii) that elevated cytosolic calcium initiates vesicle exocytosis, and (iii) that the vesicles release a factor or set of factors which depolarizes the plasmalemma and promotes ethylene synthesis. One consequence of such exocytosis should be small, extracellularly observable voltage transients. Transients, ranging in size up to 600 microvolts and possessing risetimes (10-90%) of approximately 200 ms, are known to be elicited in etiolated stems of Pisum sativum L. by friction and are here shown to be elicited by sudden increase of auxin concentration and also by a Ca2+ ionophore.

Hormonal control of meiosis: In vitro induced release of calcium ions from the plasma membrane in starfish oocytes

Using the photoprotein aequorin as a marker, we found that the Ca 2+ release, which we demonstrated to occur from the inner part of the plasma membrane after hormonal stimulation of intact starfish oocytes can also be recorded in vitro. Our acellular system was prepared from cortices of Marthasterias glacialis oocytes isolated in Ca 2+-free medium and extracted with Triton X-100. At 0–4 °C, the 100 000 g pellet obtained from this plasma membrane-rich extract responds in less than 0.1 sec to the hormone 1-methyladenine, its biological active analogues and mimetics, whereas supernatant exhibits a quite lower activity which is no longer present in the head fraction recovered after dialysis through Diaflo UM 2 filters. The in vitro calcium response is competitively inhibited by various agents which, in the same concentration range, act on meiosis reinitiation and free calcium release by the living oocyte. These and previous data are further discussed taking into account the overall chain of events which leads to the release of meiosis inhibition.