Changes In Intracellular Calcium Concentration Research Articles

Engineering a Ca⁺⁺⁺-sensitive (bio)sensor from the pore-module of a potassium channel.

Signals recorded at the cell membrane are meaningful indicators of the physiological vs. pathological state of a cell and will become useful diagnostic elements in nanomedicine. In this project we present a coherent strategy for the design and fabrication of a bio-nano-sensor that monitors changes in intracellular cell calcium concentration and allows an easy read out by converting the calcium signal into an electrical current in the range of microampere that can be easily measured by conventional cell electrophysiology apparatus.

A single-compartment model of calcium dynamics in nerve terminals and dendrites.

This introduction describes a single-compartment model of calcium dynamics that has been applied to fluorescence measurements of intracellular free calcium concentration ([Ca(2+)]i) changes in neurons. The model describes intracellular calcium handling under simplified conditions, for which analytical expressions for the amplitude and the time constants of [Ca(2+)]i changes can be explicitly derived. In particular, it reveals the dependence of the measured [Ca(2+)]i changes on the calcium indicator concentration. Applied to experimental data from small cells or subcellular compartments, the model equations have been extremely useful for obtaining quantitative information about essential parameters of Ca(2+) influx, buffering, and clearance. We illustrate also several changes that occur when the basic assumptions do not hold (e.g., when calcium diffusion, dye saturation, or kinetic effects become significant). Finally, we discuss how the changes in calcium dynamics, which are explained by the model, have been exploited for measuring properties of calcium-driven reactions, such as those regulating short-term synaptic enhancement, vesicle recycling, and adaptation.

Menthol-Induced Changes in Mesenchymal Stem Cell Differentiation

Channels from the Transient Receptor Potential (TRP) family are involved in changes in intracellular calcium concentration. TRPM8 is a member of this family expressed in different tissues that participates in the activation of signaling cascades involved in thermosensation and proliferation. Recent studies have shown that changes in intracellular calcium concentration are associated with proliferation and differentiation of mesenchymal stem cells (MSC). However, it is unknown whether TRPM8 channels are expressed in MSC and if modulation of these channels affects the proliferation and differentiation of this kind of cells. The expression of TRPM8 channels was evaluated in adipose-derived human mesenchymal stem cells that have been deeply studied due to its potential in regenerative medicine. The results obtained by RT-PCR, western blot and flow cytometry showed the presence of TRPM8 transcript and protein in adipose-derived human mesenchymal stem cells. Additionally, it was found that regulation of the TRPM8 activity by menthol although does not affect cell viability, it decreases the differentiation of MSC to adipocytes. These results strongly suggest that TRPM8 channels participate in the regulation of mesenchymal stem cells differentiation. Supported by Pontificia Universidad Javeriana, Grant ID 3937.

TRPC3 and TRPC6 Contribute to the Pathogenesis of Hypertension

Recently studies found that TRPC3 and TRPC6 played an important role in cardiovascular disease. Hypertension, as a cardiovascular disease causing the highest morbidity and mortality, has close relationship with the expressions of TRPC3 and TRPC6. Unbalanced calcium homeostasis is the major factor of pathogenesis of hypertension. Changes of intracellular calcium concentration depend on calcium transmembrane transportation, intracellular calcium store releasing and other processes. TRPC3, TRPC6 molecules, as non-selective cation channels on the cell membranes, are involved in the processes. This review illustrated the functions of TRPC3 and TRPC6 on myocardial cells, smooth muscle cells and inflammatory cells in the development of hypertension, and the effects of drugs like sildenafil to provide a new way for the prevention and treatment of hypertension.

Auranofin, an anti-rheumatic gold compound, modulates apoptosis by elevating the intracellular calcium concentration ([ca2+]I) in mcf-7 breast cancer cells.

Auranofin, a transition metal complex is used for the treatment of rheumatoid arthritis but is also an effective anti-cancer drug. We investigate the effects of Auranofin in inducing cell death by apoptosis and whether these changes are correlated to changes of intracellular calcium concentration ([Ca2+]i) in breast cancer cells (MCF-7). Cytotoxicity of Auranofin was evaluated using MTS assay and the Trypan blue dye exclusion method. With fluorescent dyes SR-FLICA and 7-AAD apoptotic death and necrotic death were differentiated by Flow cytometry. A concentration dependent decrease in the viability occurred and cells were shifted to the apoptotic phase. Intracellular calcium ([Ca2+]i) was recorded using florescence microscopy and a calcium sensitive dye (Fluo-4 AM) with a strong negative correlation (r = −0.713) to viability. Pharmacological modulators 2-APB (50 μM), Nimodipine (10 μM), Caffeine (10 mM), SKF 96365(20 μM) were used to modify calcium entry and release. Auranofin induced a sustained increase of [Ca2+]i in a concentration and time dependent manner. The use of different blockers of calcium channels did not reveal the source for the rise of [Ca2+]i. Overall, elevation of [Ca2+]i by Auranofin might be crucial for triggering Ca2+-dependent apoptotic pathways. Therefore, in anti-cancer therapy, modulating [Ca2+]i should be considered as a crucial factor for the induction of cell death in cancer cells.

An in silico case study of idiopathic dilated cardiomyopathy via a multi-scale model of the cardiovascular system

Mathematical modelling has been used to comprehend the pathology and the assessment of different treatment techniques such as heart failure and left ventricular assist device therapy in the cardiovascular field. In this study, an in-silico model of the heart is developed to understand the effects of idiopathic dilated cardiomyopathy (IDC) as a pathological scenario, with mechanisms described at the cellular, protein and organ levels. This model includes the right and left atria and ventricles, as well as the systemic and pulmonary arteries and veins.First, a multi-scale model of the whole heart is simulated for healthy conditions. Subsequently, the model is modified at its microscopic and macroscopic spatial scale to obtain the characteristics of IDC. The extracellular calcium concentration, the binding affinity of calcium binding proteins and the maximum and minimum elastances have been identified as key parameters across all relevant scales. The modified parameters cause a change in (a) intracellular calcium concentration characterising cellular properties, such as calcium channel currents or the action potential, (b) the proteins being involved in the sliding filament mechanism and the proportion of the attached crossbridges at the protein level, as well as (c) the pressure and volume values at the organ level. This model allows to obtain insight and understanding of the effects of the treatment techniques, from a physiological and biological point of view.

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca2+ concentrations in the mutant strain Aspergillus awamori 66A, containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity ofsporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 microM) stimulated spore germination: the number of germinating spores was 2-3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 microM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca2+ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 microM) were shown to cause an instantaneous increase in Ca2+ concentrations, compared to the control and lower amphotericin concentration (2.5 microM). Ca2+ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.

Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering.

The calcium-binding protein calmodulin (CaM) directly binds to membrane transport proteins to modulate their function in response to changes in intracellular calcium concentrations. Because CaM recognizes and binds to a wide variety of target sequences, identifying CaM-binding sites is difficult, requiring intensive sequence gazing and extensive biochemical analysis. Here, we describe a straightforward computational script that rapidly identifies canonical CaM-binding motifs within an amino acid sequence. Analysis of the target sequences from high resolution CaM-peptide structures using this script revealed that CaM often binds to sequences that have multiple overlapping canonical CaM-binding motifs. The addition of a positive charge discriminator to this meta-analysis resulted in a tool that identifies potential CaM-binding domains within a given sequence. To allow users to search for CaM-binding motifs within a protein of interest, perform the meta-analysis, and then compare the results to target peptide-CaM structures deposited in the Protein Data Bank, we created a website and online database. The availability of these tools and analyses will facilitate the design of CaM-related studies of ion channels and membrane transport proteins.

Role of the mitochondrial Ca2+ uniporter in Pb2+-induced oxidative stress in human neuroblastoma cells

Lead (Pb2+) has been shown to induce cellular oxidative stress, which is linked to changes in intracellular calcium (Ca2+) concentration. The mitochondrial Ca2+ uniporter (MCU) participates in the maintenance of Ca2+ homeostasis in neurons, but its role in Pb2+-induced oxidative stress is unclear. To address this question, oxidative stress was induced in human neuroblastoma SH-SY5Y cells and in newborn rats by Pb2+ treatment. The results showed that the production of reactive oxygen species is increased in cells upon treatment with Pb2+ in a dose-dependent manner, while glutathione and MCU expression were reduced. Moreover, neuronal nitric oxide synthase protein expression was elevated in rats exposed to Pb2+ during gestation, while MCU expression was decreased. Application of the MCU activator spermine or MCU overexpression reversed Pb2+-induced oxidative stress and inhibition of mitochondrial Ca2+ uptake, while the MCU inhibitor Ru360 and MCU knockdown potentiated the effects of Pb2+. These results indicate that the MCU mediates the Pb2+-induced oxidative stress response in neurons through the regulation of mitochondrial Ca2+ influx.

Abnormal intracellular calcium homeostasis associated with vulnerability in the nerve cells from heroin-dependent rat

The cellular mechanisms by which opiate addiction develops with repetitive use remain largely unresolved. Intercellular calcium homeostasis is one of the most critical elements to determine neuroadaptive changes and neuronal fate. Heroin, one of the most addictive opiates, may induce neurotoxicity potentially inducing brain impairment, especially for those chronic users who get an overdose. Here we examined changes in intracellular calcium concentration ([Ca2+]i) after repeated exposure to heroin using cultured cerebral cortical neurons. Dynamic changes in [Ca2+]i indicated by fluo-3-AM were monitored using confocal laser scan microscopy, followed by cytotoxicity assessments. It showed that the cells dissociated from heroin-dependent rats had a smaller depolarization-induced [Ca2+]i responses, and a higher elevation in [Ca2+]i when challenged with a high concentration of heroin (500μM). The restoration ability to remove calcium after washout of these stimulants was impaired. Calcium channel blocker verapamil inhibited the heroin-induced [Ca2+]i elevations as well as the heroin-induced cell damage. The relative [Ca2+]i of the nerve cells closely correlated with the number of damaged cells induced by heroin. These results demonstrate that nerve cells from heroin-dependent rats manifest abnormal [Ca2+]i homeostasis, as well as vulnerability to heroin overdose, suggesting involvement of [Ca2+]i regulation mechanisms in heroin addiction and neurotoxicity.

Analysis of the expression of eNOS in erythrocytes under hypoxic conditions (1013.15)

Background: Congenital heart diseases are among the most common birth defects, being the second leading cause of infant death in Mexico. Some of these pathologies are characterized by low concentrations of O2 in the blood, resulting in cyanosis. There is evidence that erythrocytes express active and functional eNOS, which catalyzes the production of nitric oxide (NO). NO activity in erythrocytes appears to include the regulation of membrane deformability and inhibition of platelet activation; however, the activities of eNOS, NO export to the vasculature and the relative contribution of erythrocytes to the active NO pool in circulation, have not yet been explored in detail.Objective: To identify differences in eNOS expression and activity in erythrocytes from children with congenital heart disease and cyanosis, compared to healthy controls.Methods: Analysis of eNOS expression and activity by western blot and fluorometry, in erythrocytes of patients diagnosed with congenital heart disease and blood O2 saturation below 80%, at the Children's Hospital‐IMIEM, Toluca, Mexico.Results: 30 samples from patients with congenital heart disease and blood O2 saturation less than 80%, and 6 samples from healthy controls matched for age and sex, have been collected. Erythrocytes of all samples have been isolated and changes in eNOS expression are being analyzed. Enzymatic activity is being analyzed by subjecting the erythrocytes to mechanical stress and by measuring changes in intracellular calcium concentrations and NO production.

The Effect of Gentian Violet on Human Anterior Lens Epithelial Cells

Purpose: To evaluate whether the gentian violet staining of the anterior lens capsule during the cataract surgery is cytotoxic for the human lens epithelial cells, as an indirect indication of possible toxicity towards the corneal endothelium and the safety of gentian violet application.Materials and methods: Two groups of anterior lens capsules obtained during the cataract surgery, gentian violet stained and non-stained, were incubated with fluorescent dye Fura-2. Their fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in free intracellular calcium concentration ([Ca2+]i) in response to pharmacological stimulation by acetylcholine. The [Ca2+]i homeostasis is the indicator of cellular function. The changes in [Ca2+]i were compared between the two groups.Results: Epithelial cells responded to acetylcholine in both groups of capsules - gentian violet stained (n = 17) and non-stained ones (n = 33). No significant differences of the elicited responses were found in rise time (p = 0.89), decay time (p = 0.61) or amplitude of [Ca2+]i (p = 0.96 for 63× and p = 0.26 for 40× objectives) between the two groups of capsules (Student t test).Conclusions: The staining of the anterior lens capsule with gentian violet during phacoemulsification in concentration of 0.01%, does not have detectable cytotoxic effects, which would affect the [Ca2+]i homeostasis in lens epithelial cells. The data, if extrapolated to corneal endothelium, exposed to the same concentration, suggest that gentian violet in concentration of 0.01% is safe as an adjunct for capsule visualization in cataract surgery.

Modeling of progesterone-induced intracellular calcium signaling in human spermatozoa

Calcium ion is a secondary messenger of mammalian spermatozoa. The dynamic change of its concentration plays a vital role in the process of sperm motility, capacitation, acrosome and fertilization. Progesterone released by the cumulus cells, as a potent stimulator of fertilization, can activate the calcium channels on the plasma membrane, which in turn triggers the dynamic change of intracellular calcium concentration. In this paper, a mathematical model of calcium dynamic response in mammalian spermatozoa induced by progesterone is proposed and numerical simulation of the dynamic model is conducted. The results show that the dynamic response of calcium concentration predicted by the model is in accordance with experimental evidence. The proposed dynamic model can be used to explain the phenomena observed in the experiments and predict new phenomena to be revealed by experimental investigations, which will provide the basis to quantitatively investigate the fluid mechanics and biochemistry for the sperm motility induced by progesterone.

600 ns pulse electric field-induced phosphatidylinositol4,5-bisphosphate depletion

The interaction between nsPEF-induced Ca2+ release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca2+ the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca2+ (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca2+ is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca2+ leads to a whole cell response. The reported observations of Ca2+ dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.

Stimulatory Effects of Glucose and Porphyromonas gingivalis Lipopolysaccharide on the Secretion of Inflammatory Mediators From Human Macrophages

Hyperglycemia is widely considered to be the causal link between diabetes mellitus (DM) and diabetic complications. The purpose of this study is to determine the effects of high glucose in the presence of lipopolysaccharide (LPS) purified from the periodontal pathogen Porphyromonas gingivalis on human macrophages. Macrophages (U937) were treated with various concentrations of P. gingivalis-LPS under normal (5.5 mM) or high (25 mM) glucose conditions. Mitochondrial dehydrogenase activity was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. The levels of inflammatory mediators secreted were determined using the enzyme-linked immunosorbent assay and the competitive enzyme immunoassay. The intracellular calcium chelator was used to examine whether the intracellular calcium was involved. Statistical differences were assessed using a one-way analysis of variance and Tukey multiple-comparison intervals with α = 0.05. High glucose condition enhanced the mitochondrial dehydrogenase activity in macrophages. P. gingivalis-LPS induced the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and prostaglandin E(2) (PGE(2)) in a dose-dependent manner both in normal and high glucose conditions. The stimulatory effects by P. gingivalis-LPS were more evident when cells were cultured under high glucose conditions. Changes of intracellular calcium concentration were involved not only in high glucose-induced mitochondrial dehydrogenase activity but also in P. gingivalis-LPS-induced production of IL-6, TNF-α, or PGE(2), especially under the high glucose conditions. High glucose appeared to enhance the inflammatory response induced by the periodontal pathogen. The information generated may help to delineate the possible mechanisms by which hyperglycemia compromises the periodontal health of patients with DM.

Analysis of calcium signals in steering neuronal growth cones in vitro.

Calcium imaging allows us to measure the spatial and temporal changes in intracellular calcium concentration in living cells. Localized calcium elevation often functions as the polarizing signal during guided migration including axon guidance. In this chapter, we describe a protocol to quantitatively monitor the spatiotemporal dynamics of calcium signals in neuronal growth cones in the presence of an extracellular concentration gradient of axon guidance cue.

Role of Mitofusin-2 in High Mobility Group Box-1 Protein-Mediated Apoptosis of T Cells in Vitro

Background: High mobility group box-1 protein (HMGB1), a ubiquitous nuclear protein, which is recognized as a danger-associated molecular pattern (DAMP) triggering activation of the innate immune system. Previous studies have shown that HMGB1 also plays a role in T cell-mediated immunity, but the effect of HMGB1 on apoptosis of T cells and its precise mechanism remain to be determined. Methods: Two kinds of apoptosis assay techniques were used, i.e., Annexin V-FITC conjunction with PI to identify early apoptotic cells, Hoechst 33342 staining for double-stranded DNA to observe nuclear fragmentation or apoptotic body. The activation status of caspase-3, caspase-8, as well as caspase-9 was examined by colorimetric assay. The dynamic changes in intracellular calcium concentration ([Ca²⁺]_i) was monitored by flow cytometry. Overexpression of Mfn2 was preformed by lentiviral vector transfection. The mRNA and protein levels of Mfn2 were determined by RT-PCR and Western-blotting. Results: Treatment of Jurkat T cells with recombinant human HMGB1 (rhHMGB1) causes a significant dose-dependent increase in percentage of apoptotic cells. When T cells are incubated with HMGB1 they express decreased mitochondria fusion-related protein mitofusin-2 (Mfn2) and activate mitochondrial apoptotic pathway via elevation of [Ca²⁺]_i, Bax insertion, and activation of caspase. Furthermore, overexpression of Mfn2 ameliorates the apoptosis of T cells induced by HMGB1. This occurs at least partly through Mfn2 keeps Ca²⁺ homeostasis in T cells evidenced by monitoring [Ca²⁺]_i dynamics. Conclusion: HMGB1 can trigger apoptosis of T lymphocytes through mitochondrial death pathway associated with [Ca²⁺]_i elevation. Mfn2 plays a pivotal role in this process, and it might be a novel therapeutic target in T cell apoptosis related disorders.

Regulating Effect of Tea Polyphenols on Endothelin, Intracellular Calcium Concentration, and Mitochondrial Membrane Potential in Vascular Endothelial Cells Injured by Angiotensin II

Polyphenols are the main active component of tea and are considered an antiatherosclerosis agent, protecting vascular endothelial cells (VECs) from injury and preventing cardiovascular diseases. Endothelin level, intracellular calcium concentration, and mitochondrial membrane potential in VECs directly reflect the function and injury status of cells. The objective of this study was to study the regulating effects of tea polyphenols on these factors in VECs injured by angiotensin II (Ang-II) and explore the protective effect of tea polyphenols on human VECs. In this study, human aortic vascular endothelial cells were divided into 4 groups: (1) a control group; (2) Ang-II group: Ang-II at a concentration of 10(-7) mol/L was added to the cells; (3) low-concentration tea polyphenols + Ang-II group: tea polyphenols at a concentration of 5 mg/L was added in addition to Ang-II; (4) high-concentration tea polyphenols + Ang-II group: tea polyphenols at a concentration of 25 mg/L was added in addition to Ang-II. One hundred microliters of supernatant was extracted before treatment and at 0.5, 6, and 24 hours after treatment in each group to establish the content of endothelin. The results showed the following: (1) tea polyphenols decreased the expression of endothelin-1 messenger RNA, which was increased by Ang-II (P < 0.01). (2) Tea polyphenols inhibited endothelin secretion induced by Ang-II (P < 0.01), and the inhibition of low-concentration tea polyphenols was superior to that of high concentration tea polyphenols (P < 0.01). (3) Tea polyphenols ameliorated the changes in intracellular calcium concentration and mitochondrial membrane potential induced by Ang-II (P < 0.01). This study suggests that tea polyphenols may effectively protect VECs against injury by regulating endothelin, intracellular calcium concentration, and mitochondrial membrane potential in VECs injured by Ang-II. Additionally, lower dose would be used clinically rather than the higher dose for obtaining better results.

Protective Effect of Metformin Against Cisplatin-Induced Ototoxicity in an Auditory Cell Line

Metformin, an antidiabetic drug with potent anticancer activity, is known to prevent oxidative stress-induced cell death in several cell types through a mechanism dependent on the mitochondria. In the present study, we investigated the influence of metformin on cisplatin ototoxicity in an auditory cell line. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (Sigma, St. Louis, MO, USA) cell proliferation assay. Oxidative stress and apoptosis were assessed by flow cytometry analysis, Hoechst 33258 staining, reactive oxygen species (ROS) measurement, and western blotting. Intracellular calcium concentration changes were detected using calcium imaging. Pretreatment with 1 mM metformin prior to the application of 20 μM cisplatin significantly decreased the frequency of late apoptosis in HEI-OC1 cells and also significantly attenuated the cisplatin-induced increase in ROS. In addition, metformin inhibited the activation of caspase-3 and levels of poly-ADP-ribose polymerase (PARP). Pretreatment with metformin prevented the cisplatin-induced elevation in intracellular calcium concentrations. We propose that metformin protects against cisplatin-induced ototoxicity by inhibiting the increase in intracellular calcium levels, preventing apoptosis, and limiting ROS production.

Uncaging Calcium in Neurons

Changes in intracellular free calcium concentration (Δ[Ca(2+)]i) driving physiological events such as neurotransmitter release or Ca(2+)-dependent currents can be monitored using Ca(2+)-sensitive fluorescent dyes. Although these dyes can correlate Δ[Ca(2+)]i with a physiological event, they cannot directly test for causality between changes in [Ca(2+)]i and that event. Photolabile Ca(2+) chelators are Ca(2+)-binding molecules that can alter and, to a certain extent, control [Ca(2+)]i in an inducible manner and with temporal and spatial resolution that surpasses microinjection or ionophore application. Here we discuss the properties of caged Ca(2+) compounds as well as some practical considerations for their use in neuronal cells, where they have proven particularly effective.