Endogenous Calcium Research Articles

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We developed SpiCee, a versatile and genetically-encoded chelator combining low and high affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically-controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

β-blockers augment L-type Ca2+ channel activity by targeting spatially restricted β2AR signaling in neurons.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of β-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate β2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated β2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant β2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant β2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which β-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Hypocalcemia and Massive Blood Transfusions: A Pilot Study in a Level I Trauma Center.

Trauma is a leading cause of death in the United States, and uncontrolled hemorrhage is often the primary cause of mortality. Massive transfusions provide lifesaving treatment for the bleeding trauma patient; yet, this is not a benign intervention. Calcium levels can be significantly decreased with rapidly transfused blood products due to the citrate preservative that is added. Citrate binds to the patient's endogenous calcium when blood products are administered, rendering calcium inactive. As a result, undesirable physiological effects can occur. Although there is a plethora of evidence reporting the negative effects of hypocalcemia during resuscitation, the research for standardization of calcium monitoring and replacement during a massive transfusion event is less robust. Consequently, monitoring and replacement of this vital electrolyte are often overlooked. Trauma department employees at an urban academic hospital were given a pretest to assess their knowledge of calcium monitoring and replacement during a massive transfusion. On the basis of test results and a need for staff education, a short, animated video was designed and distributed for viewing. Following the educational video, a posttest was administered and yielded higher scores when compared with the pretest (p = .001). Lack of knowledge and national standards may be root causes for hypocalcemia. Educational interventions such as innovative, brief videos can be effective for enhancing staff members' knowledge and improving patient care.

Laboratory method of microbial induced solidification/stabilization for municipal solid waste incineration fly ash

This paper presents a method to solidify/stabilize the municipal solid waste incineration (MSWI) fly ash by originally employing the microbial induced carbonate precipitation (MICP) technique. In this method, the rich endogenous calcium in the MSWI fly ash was utilized to induce calcite precipitation, which is different from the operation of adding extra calcium source in previous researches. The fly ash sample had a CaO content of 44.5%, and its leaching concentrations of Zn, Cr and Pb exceed the limits of the identification standard for hazardous wastes in China. The optical density at 600 nm (OD600) of the bacterial solution was about 1.0 after the processes of bacterial activation and reproduction. The prepared fly ash sample was well mixed with bacterial solution at an ash-liquid ratio of 1 kg: 0.3 L and cured at a temperature of 20 °C and a humidity of ≥95% for 7 days. After treatment, the heavy metal leachability significantly reduced to meet the standard for pollution control of landfill site, and the unconfined compressive strength increased approximately 40%. The precipitated carbonates were verified by SEM-EDS analysis and quantified by measurement of carbonate content via acid-dissolving method. The results shone a light on the possibility of using MICP technique as a useful and efficient tool to stabilize the MSWI fly ash before being reused or properly stored in landfills.•The MICP method is efficient to reduce the heavy metal leachability and increase the compressive strength of MSWI fly ash.•The endogenous calcium in MSWI fly ash was utilized to induce calcite precipitation.•The heavy metals in MSWI fly ash were well immobilized by the formation of carbonates.

Single-Cell Transcriptomics of Human Oocytes: Environment-Driven Metabolic Competition and Compensatory Mechanisms During Oocyte Maturation.

Aims: The mechanisms coordinating maturation with an environment-driven metabolic shift, a critical step in determining the developmental potential of human in vitro maturation (IVM) oocytes, remain to be elucidated. Here we explored the key genes regulating human oocyte maturation using single-cell RNA sequencing and illuminated the compensatory mechanism from a metabolic perspective by analyzing gene expression.Results: Three key genes that encode CoA-related enzymes were screened from the RNA sequencing data. Two of them, ACAT1 and HADHA, were closely related to the regulation of substrate production in the Krebs cycle. Dysfunction of the Krebs cycle was induced by decreases in the activity of specific enzymes. Furthermore, the activator of these enzymes, the calcium concentration, was also decreased because of the failure of influx of exogenous calcium. Although release of endogenous calcium from the endoplasmic reticulum and mitochondria met the requirement for maturation, excessive release resulted in aneuploidy and developmental incompetence. High nicotinamide nucleotide transhydrogenase expression induced NADPH dehydrogenation to compensate for the NADH shortage resulting from the dysfunction of the Krebs cycle. Importantly, high NADP+ levels activated DPYD to enhance the repair of DNA double-strand breaks to maintain euploidy.Innovation: The present study shows for the first time that exposure to the in vitro environment can lead to the decline of energy metabolism in human oocytes during maturation but that a compensatory action maintains their developmental competence.Conclusion: In vitro maturation of human oocytes is mediated through a cascade of competing and compensatory actions driven by genes encoding enzymes.

Ablation of α2δ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo

The auxiliary α2δ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, α2δ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of α2δ-1 on CaV2.2 localization in the pain pathway in vivo, where CaV2.2 is important for nociceptive transmission and α2δ-1 plays a critical role in neuropathic pain. We find CaV2.2 is preferentially expressed on the plasma membrane of calcitonin gene-related peptide-positive small nociceptors. This is paralleled by strong presynaptic expression of CaV2.2 in the superficial spinal cord dorsal horn. EM-immunogold localization shows CaV2.2 predominantly in active zones of glomerular primary afferent terminals. Genetic ablation of α2δ-1 abolishes CaV2.2 cell-surface expression in dorsal root ganglion neurons and dramatically reduces dorsal horn expression. There was no effect of α2δ-1 knockout on other dorsal horn pre- and postsynaptic markers, indicating the primary afferent pathways are not otherwise affected by α2δ-1 ablation.

Endogenous calcium attenuates the immunomodulatory activity of a polysaccharide from Lycium barbarum L. leaves by altering the global molecular conformation

A heteropolysaccharide, LP5, was purified from Lycium barbarum L. leaves. It was identified as a calcium-rich polysaccharide (8.6 mg calcium/g) with a molecular weight of 2.50 × 105 Da. The polysaccharide was composed of six kinds of monosaccharides, of which mannose and xylose are the main components. And it contains 16.37% glucuronic acid. Studies on RAW264.7 cells demonstrated that this polysaccharide exhibited potent immunomodulatory activity, including an increase in phagocytic activity, as well as the release of both nitric oxide and cytokines. However, after the depletion of calcium, the polysaccharide exhibited greater immunomodulatory activity (p < 0.05). Further conformation analysis confirmed that the polysaccharide changed from a compact spherical conformation to a random coil structure in aqueous solution after the depletion of calcium, which resulted in increased immunomodulatory activity by LP5.

Novel Bivalent and D-Peptide Ligands of CXCR4 Mobilize Hematopoietic Progenitor Cells to the Blood in C3H/HeJ Mice

The interaction of SDF-1α (also known as CXCL12) with the CXCR4 receptor plays a critical role in the retention of hematopoietic stem cells (HSCs) in bone marrow. The viral macrophage inflammatory protein-II (vMIP-II), a human herpesvirus-8 (HHV-8)-encoded viral chemokine, can bind the CXCR4 receptor and inhibit endogenous ligand-induced calcium responses and cell migration. Previously, we used the bivalent ligand approach to link synthetically two unnatural D-amino acid peptides derived from the N-terminus of vMIP-II (DV1 and DV3, respectively) to generate a dimeric peptide, DV1-K-(DV3) (also named HC4319), which shows very high affinity for CXCR4. Here, we studied the biological effects of this dimeric peptide, HC4319, and its monomeric counterpart, DV1, on SDF-1α-induced signaling in CXCR4- or CXCR7-transfected Chinese hamster ovary cells and mobilization of hematopoietic progenitor cells (HPCs) in C3H/HeJ mice using an HPC assay. HC4319 and DV1 inhibited significantly the phosphorylation of Akt and Erk, known to be downstream signaling events of CXCR4. This in vivo study in C3H/HeJ mice showed that HC4319 and DV-1 strongly induced rapid mobilization of granulocyte–macrophage colony-forming units (CFUs), erythrocyte burst-forming units, and granulocyte–erythrocyte–monocyte–megakaryocyte CFUs from the bone marrow to the blood. These results provide the first reported experimental evidence that bivalent and D-amino acid peptides derived from the N-terminus of vMIP-II are potent mobilizers of HPCs in C3H/HeJ mice and support the further development of such agents for clinical application.

Homer 1a Induces Calcium Channel Activation, but Does Not Change Their Properties in A431 Cells.

Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells. Monomeric Homer 1a proteins increase activity of Imin channels, but did not modulate their electrophysiological properties. Recombinant Homer 1c protein did not block the induced calcium currents.

Methodological study on endogenous calcium absorptivity using rats and 41Ca tracing

Elemental calcium plays an important role in human physiology. In order to study the relationship between Ca-intake, Ca-chemical formulation, and Ca-absorptivity, a balance experiment using a 41Ca tracer technique in SD rats was conducted to measure the endogenous fecal calcium and true absorption of calcium. Apparent absorption of calcium was measured as a control to the endogenous calcium labeling experiment. These results show that by using 41Ca labeled endogenous calcium in vivo, researchers could obtain the true calcium absorption data without extrinsic labeling. Therefore, the method was not affected by the chemical structure or type of calcium supplement and might be used in evaluating the absorptivity of marketed calcium supplements.

Peripheral nerve injury increases contribution of L-type calcium channels to synaptic transmission in spinal lamina II: Role of α2δ-1 subunits.

BackgroundFollowing peripheral nerve chronic constriction injury, the accumulation of the α2δ–1 auxiliary subunit of voltage-gated Ca2+ channels in primary afferent terminals contributes to the onset of neuropathic pain. Overexpression of α2δ–1 in Xenopus oocytes increases the opening properties of Cav1.2 L-type channels and allows Ca2+ influx at physiological membrane potentials. We therefore posited that L-type channels play a role in neurotransmitter release in the superficial dorsal horn in the chronic constriction injury model of neuropathic pain.ResultsWhole-cell recording from lamina II neurons from rats, subject to sciatic chronic constriction injury, showed that the L-type Ca2+ channel blocker, nitrendipine (2 µM) reduced the frequency of spontaneous excitatory postsynaptic currents. Nitrendipine had little or no effect on spontaneous excitatory postsynaptic current frequency in neurons from sham-operated animals. To determine whether α2δ–1 is involved in upregulating function of Cav1.2 L-type channels, we tested the effect of the α2δ–1 ligand, gabapentin (100 µM) on currents recorded from HEK293F cells expressing Cav1.2/β4/α2δ–1 channels and found a significant decrease in peak amplitude with no effect on control Cav1.2/β4/α2δ–3 expressing cells. In PC-12 cells, gabapentin also significantly reduced the endogenous dihydropyridine-sensitive calcium current. In lamina II, gabapentin reduced spontaneous excitatory postsynaptic current frequency in neurons from animals subject to chronic constriction injury but not in those from sham-operated animals. Intraperitoneal injection of 5 mg/kg nitrendipine increased paw withdrawal threshold in animals subject to chronic constriction injury.ConclusionWe suggest that L-type channels show an increased contribution to synaptic transmission in lamina II dorsal horn following peripheral nerve injury. The effect of gabapentin on Cav1.2 via α2δ–1 may contribute to its anti-allodynic action.

Deletion of Orai2 augments endogenous CRAC currents and degranulation in mast cells leading to enhanced anaphylaxis

All three members of the Orai family of cation channels–Orai1, Orai2 and Orai3–are integral membrane proteins that can form store-operated Ca2+ channels resembling endogenous calcium release-activated channels (CRAC) in many aspects. Loss of function studies in human and murine models revealed many functions of Orai1 proteins not only for Ca2+ homeostasis, but also for cellular and systemic functions in many cell types. By contrast, the knowledge regarding the contribution of Orai2 and Orai3 proteins in these processes is sparse. In this study, we report the generation of mouse models with targeted inactivation of the Orai2 gene to study Orai2 function in peritoneal mast cells (PMC), a classical cell model for CRAC channels and Ca2+-dependent exocytosis of inflammatory mediators. We show that the Ca2+ rise triggered by agonists acting on high-affinity Fc receptors for IgE or on MAS-related G protein-coupled receptors is significantly increased in Orai2-deficient mast cells. Ca2+ entry triggered by depletion of intracellular stores (SOCE) is also increased in Orai2−/− PMCs at high (2mM) extracellular Ca2+ concentration, whereas SOCE is largely reduced upon re-addtion of lower (0.1mM) Ca2+ concentration. Likewise, the density of CRAC currents, Ca2+-dependent mast cell degranulation, and mast cell-mediated anaphylaxis are intensified in Orai2-deficient mice. These results show that the presence of Orai2 proteins limits receptor-evoked Ca2+ transients, store-operated Ca2+ entry (SOCE) as well as degranulation of murine peritoneal mast cells but also raise the idea that Orai2 proteins contribute to Ca2+ entry in connective tissue type mast cells in discrete operation modes depending on the availability of calcium ions in the extracellular space.

Supplementation of pig diets in the growth and termination phases with different calcium sources.

The aim of this study was to evaluate the effect of supplementation of pig diets in the growth and termination phases with different calcium sources. In experiment I, 36 whole males were distributed in randomized blocks in six groups, with six replications. A basal diet was formulated to meet the animals' nutritional requirements except for calcium (0.09%), and the sources evaluated (calcitic limestone, monodicalcium phosphate, calcinated bone flour, and oyster flour) replaced the basal diet to provide 0.59% of total calcium. To determine the endogenous calcium, a diet containing low calcium (0.019%) was given simultaneously to another group of animals. Feces and urine were collected for determination the coefficients of apparent and true digestibility. In experiment II, 160 piglets were distributed in randomized blocks in four treatments, with five replications and four animals per experimental unit. Carcass and performance parameters, calcium concentration in bone and serum, and bone parameters were evaluated. The data were submitted to analysis of variance and factorial. The calcium source did not influence the digestibility coefficients determined by total collection (P>0.05). The digestibility of Ca from oyster flour estimated by collection with an indicator was higher than that from the other sources (P<0.05). Calcium sources did not interfere in the evaluated parameters (P>0.05). The sources studied in this work can be used to supplement growing pigs' diets.

Knockdown of amyloid precursor protein increases calcium levels in the endoplasmic reticulum

Familial Alzheimer’s disease (AD) is caused by mutations in the genes that encode amyloid precursor protein (APP) and presenilins. Disturbances in calcium homeostasis have been observed in various cellular and animal models of AD and are proposed to underlie the pathogenesis of the disease. Furthermore, wildtype presenilins were shown to regulate endoplasmic reticulum (ER) calcium homeostasis, although their precise mechanism of action remains controversial. To investigate whether APP also affects ER calcium levels, we used RNA interference to target the APP gene in cultured T84 cells in combination with two types of ER calcium sensors. Using a genetically encoded calcium indicator, GEM-CEPIA1er, we found that APP-deficient cells exhibited elevated resting calcium levels in the ER and prolonged emptying of ER calcium stores upon the cyclopiazonic acid-induced inhibition of sarco-endoplasmic reticulum calcium-ATPase. These effects could be ascribed to lower ER calcium leakage rates. Consistent with these results, translocation of the endogenous ER calcium sensor STIM1 to its target channel Orai1 was delayed following ER calcium store depletion. Our data suggest a physiological function of APP in the regulation of ER calcium levels.

Polysaccharide from Lycium barbarum L. leaves enhances absorption of endogenous calcium, and elevates cecal calcium transport protein levels and serum cytokine levels in rats

A high-mineral polysaccharide (LP) was extracted from Lycium barbarum L. leaves, and the absorption of endogenous calcium, iron, and zinc was evaluated in Caco-2 cells and rat models. The absorption of these minerals ranged from 2.7 to 5.9% in Caco-2 cells, compared with 60.5–81.9% in rat models. LP had a compact globular conformation at pH∼7.2 when co-cultured with Caco-2 cells, and degraded to a loosely linear structure at pH∼6.7 in rat cecum. The degraded polysaccharides didn’t change the mucosal morphometry but caused higher expression of calcium transporter 1 (CAT1) and zinc transporter 1 (ZIP1) proteins in rat cecum. Furthermore, fermentation of polysaccharides in cecum also caused elevated levels of serum cytokines, including TNF-α and IL-6. GL polysaccharides could increase the bioavailability of endogenous calcium via changes in the molecular conformation and through upregulation of the expression of mineral transporter proteins.

Ultrafast Near-Infrared Light-triggered Intracellular Uncaging to Probe Cell Signaling.

The possibility of regulating cell signaling with high spatial and temporal resolution within individual cells and complex cellular networks has important implications in biomedicine. In this report, we demonstrate a general strategy that uses near-infrared tissue-penetrating laser pulses to uncage biomolecules from plasmonic gold-coated liposomes, i.e. plasmonic liposomes, to activate cell signaling in a non-thermal, ultrafast and highly controllable fashion. Near-infrared picosecond laser pulse induces transient nanobubbles around plasmonic liposomes. The mechanical force generated from the collapse of nanobubbles rapidly ejects encapsulated compound within 0.1 ms. We showed that single pulse irradiation triggers the rapid intracellular uncaging of calcein from plasmonic liposomes inside endo-lysosomes. The uncaged calcein then evenly distributes over the entire cytosol and nucleus. Furthermore, we demonstrated the ability to trigger calcium signaling in both an immortalized cell line and primary dorsal root ganglion (DRG) neurons by intracellular uncaging of inositol triphosphate (IP3), an endogenous cell calcium signaling second messenger. Compared with other uncaging techniques, this ultrafast near-infrared light-driven molecular uncaging method is easily adaptable to deliver a wide range of bioactive molecules with an ultrafast optical switch, enabling new possibilities to investigate signaling pathways within individual cells and cellular networks.

29: CEILIAC DISEASE AND BONE MASS DENSITY

Background and aims:Celiac disease (CD), an autoimmune disease initiated by the ingestion of food containing gluten. Constant intolerance to gluten cause damages of the small intestinal mucosa. One of reasons...

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors.

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

Endogenous and Exogenous Calcium Involved in the Betulin Production from Submerged Culture of Phellinus linteus Induced by Hydrogen Sulfide.

Using pharmacological and biochemical approaches, Ca(2+) involved in the betulin production in mycelia of Phellinus linteus induced by hydrogen sulfide (H2S) were investigated. The results showed that 2 mM H2S donor NaHS or 10 mM CaCl2 was found to enhance the betulin content in the mycelia of Phellinus to the maximum, which were 112.43 and 93.24% higher than that in the control, respectively. Further, NaHS and CaCl2 co-treatment also showed positive outcome, which were 128.95 or 24.52% higher than that in the control or NaHS treatment. At the same time, NaHS also enhanced the content of Ca(2+) and CaM. But, the above positive inductive effects for Ca(2+), CaM, and betulin production can be blocked with either Ca(2+) channel blocker (LaCl3, 2-aminoethoxydiphenyl borate) or Ca(2+) chelator (ethylenediaminetetraacetic acid (EDTA)). Among of them, betulin content was reduced 35.06% by NaHS and EGTA to the minimum, and this reduction could be reversed by the application of CaCl2 (NaHS + EGTA + CaCl2). From above results, it can be concluded that endogenous and exogenous calcium involved in the betulin production from submerged culture of P. linteus induced by hydrogen sulfide.

Yhhu4488, a novel GPR40 agonist, promotes GLP-1 secretion and exerts anti-diabetic effect in rodent models

G protein-coupled receptor 40 (GPR40) is predominantly expressed in pancreatic β-cells and activated by long-chain fatty acids. GPR40 has drawn considerable interest as a potential therapeutic target for type 2 diabetes mellitus (T2DM) due to its important role in enhancing glucose-stimulated insulin secretion (GSIS). Encouragingly, GPR40 is also proven to be highly expressed in glucagon-like peptide-1 (GLP-1)-producing enteroendocrine cells afterwards, which opens a potential role of GPR40 in enhancing GLP-1 secretion to exert additional anti-diabetic efficacy. In the present study, we discovered a novel GPR40 agonist, yhhu4488, which is structurally different from other reported GPR40 agonists. Yhhu4488 showed potent agonist activity with EC50 of 49.96 nM, 70.83 nM and 58.68 nM in HEK293 cells stably expressing human, rat and mouse GPR40, respectively. Yhhu4488 stimulated GLP-1 secretion from fetal rat intestinal cells (FRIC) via triggering endogenous calcium store mobilization and extracellular calcium influx. The effect of yhhu4488 on GLP-1 secretion was further confirmed in type 2 diabetic db/db mice. Yhhu4488 exhibited satisfactory potency in in vivo studies. Single administration of yhhu4488 improved glucose tolerance in SD rats. Chronic administration of yhhu4488 effectively decreased fasting blood glucose level, improved β-cell function and lipid homeostasis in type 2 diabetic ob/ob mice. Taken together, yhhu4488 is a novel GPR40 agonist that enhances GLP-1 secretion, improves metabolic control and β-cell function, suggesting its promising potential for the treatment of type 2 diabetes.