Extracellular Calcium Ion Concentration Research Articles

A light-addressable potentiometric sensor-based extracellular calcium dynamic monitoring and imaging platform for cellular calcium channel drug evaluation

Disruption and dysregulation of cellular calcium channel function can lead to diseases such as ischemic stroke, heart failure, and arrhythmias. Corresponding calcium channel drugs typically require preliminary efficacy evaluations using in vitro models such as cells and simulated tissues before clinical testing. However, traditional detection and evaluation methods often encounter challenges in long-term continuous monitoring and lack calcium specificity. In this study, a dynamic monitoring system based on ion-sensitive membranes for light-addressable potentiometric sensor (LAPS) was developed to meet the demand for monitoring changes in extracellular calcium ion (Ca2+) concentration in live cells. The effects of Ca2+ channel agonists and blockers on 2D and 3D HL-1 cells were investigated, with changes in extracellular Ca2+ concentration reflecting cellular calcium metabolism, facilitating drug evaluation. Additionally, calcium imaging technology with optical addressing capability complemented the LAPS system's ability to perceive 3D cell morphology, enhancing its drug evaluation capabilities. This work provides a novel, label-free, specific, and stable technique for monitoring cellular calcium metabolism. It achieves both continuous monitoring at single points and custom sensing area calcium imaging, holding significant implications for drug screening and disease treatment related to human calcium homeostasis.

Evaluation of mechanisms involved in regulation of intrinsic excitability by extracellular calcium in CA1 pyramidal neurons of rat.

It is well recognized that changes in the extracellular concentration of calcium ions influence the excitability of neurons, yet what mechanism(s) mediate these effects is still a matter of debate. Using patch-clamp recordings from rat hippocampal CA1 pyramidal neurons, we examined the contribution of G-proteins and intracellular calcium-dependent signaling mechanisms to changes in intrinsic excitability evoked by altering the extracellular calcium concentration from physiological (1.2 mM) to a commonly used experimental (2 mM) level. We find that the inhibitory effect on intrinsic excitability of calcium ions is mainly expressed as an increased threshold for action potential firing (with no significant effect on resting membrane potential) that is not blocked by either the G-protein inhibitor GDPβS or the calcium chelator BAPTA. Our results therefore argue that in the concentration range studied, G-protein coupled calcium-sensing receptors, non-selective cation conductances, and intracellular calcium signaling pathways are not involved in mediating the effect of extracellular calcium ions on intrinsic excitability. Analysis of the derivative of the action potential, dV/dt versus membrane potential, indicates a current shift towards more depolarized membrane potentials at the higher calcium concentration. Our results are thus consistent with a mechanism in which extracellular calcium ions act directly on the voltage-gated sodium channels by neutralizing negative charges on the extracellular surface of these channels to modulate the threshold for action potential activation.

The Effect of Calcium Ions on the Electrophysiological Properties of Single ANO6 Channels.

Proteins belonging to the anoctamin (ANO) family form calcium-activated chloride channels (CaCCs). The most unusual member of this family, ANO6 (TMEM16F), simultaneously exhibits the functions of calcium-dependent scramblase and the ion channel. ANO6 affects the plasma membrane dynamics and phosphatidylserine transport; it is also involved in programmed cell death. The properties of ANO6 channels remain the subject of debate. In this study, we investigated the effect of variations in the intracellular and extracellular concentrations of calcium ions on the electrophysiological properties of endogenous ANO6 channels by recording single ANO6 channels. It has been demonstrated that (1) a high calcium concentration in an extracellular solution increases the activity of endogenous ANO6 channels, (2) the permeability of endogenous ANO6 channels for chloride ions is independent of the extracellular concentration of calcium ions, (3) that an increase in the intracellular calcium concentration leads to the activation of endogenous ANO6 channels with double amplitude, and (4) that the kinetics of the channel depend on the plasma membrane potential rather than the intracellular concentration of calcium ions. Our findings give grounds for proposing new mechanisms for the regulation of the ANO6 channel activity by calcium ions both at the inner and outer sides of the membrane.

Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1–100 μM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

A Silicon-Based Field-Effect Biosensor for Drug-Induced Cardiac Extracellular Calcium Ion Change Detection.

Calcium ions participate in the regulation of almost all biological functions of the body, especially in cardiac excitation-contraction coupling, acting as vital signaling through ion channels. Various cardiovascular drugs exert their effects via affecting the ion channels on the cell membrane. The current strategies for calcium ion monitoring are mainly based on fluorescent probes, which are commonly used for intracellular calcium ion detection (calcium imaging) and cannot achieve long-term monitoring. In this work, an all-solid-state silicone-rubber ion-sensitive membrane was fabricated on light-addressable potentiometric sensors to establish a program-controlled field-effect-based ion-sensitive light-addressable potentiometric sensor (LAPS) platform for extracellular calcium ion detection. L-type calcium channels blocker verapamil and calcium channel agonist BayK8644 were chosen to explore the effect of ion channel drugs on extracellular calcium ion concentration in HL-1 cell lines. Simultaneously, microelectrode array (MEA) chips were employed to probe the HL-1 extracellular field potential (EFP) signals. The Ca2+ concentration and EFP parameters were studied to comprehensively evaluate the efficacy of cardiovascular drugs. This platform provides more dimensional information on cardiovascular drug efficacy that can be utilized for accurate drug screening.

Ethylenediaminetetraacetic acid (EDTA) enhances cAMP production in human TDAG8-expressing cells

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds tightly to metal ions. We found that cAMP response element (CRE)-driven promoter activity by protons was enhanced by EDTA in human T-cell death-associated gene 8 (TDAG8)-overexpressed HEK293T cells. The enhancing action by EDTA was also detected by proton-induced cAMP production that is located upstream from the CRE-driven promoter activity even at physiological proton concentration pH7.4. The proton-induced CRE-driven promoter activity was not enhanced by other chelating agents, ethylene glycol tetraacetic acid (EGTA) and sodium citrate. The enhanced CRE-driven promoter activity by EDTA was not attenuated by increasing the extracellular calcium ion concentration. These results indicate that the EDTA-enhancing action may not be due to its chelating action but might rather be another EDTA-specific effect. Enhanced cAMP production by EDTA was also detected in a human leukemia cell line HL-60, in which TDAG8 and OGR1 (ovarian cancer G-protein-coupled receptor 1) were endogenously expressed, suggesting that the medical use of EDTA would influence the physiological and pathophysiological functions of hematopoietic cells.

Quantification of bone marrow interstitial pH and calcium concentration by intravital ratiometric imaging

The fate of hematopoietic stem cells (HSCs) can be directed by microenvironmental factors including extracellular calcium ion concentration ([Ca2+]e), but the local [Ca2+]e around individual HSCs in vivo remains unknown. Here we develop intravital ratiometric analyses to quantify the absolute pH and [Ca2+]e in the mouse calvarial bone marrow, taking into account the pH sensitivity of the calcium probe and the wavelength-dependent optical loss through bone. Unexpectedly, the mean [Ca2+]e in the bone marrow (1.0 ± 0.54 mM) is not significantly different from the blood serum, but the HSCs are found in locations with elevated local [Ca2+]e (1.5 ± 0.57 mM). With aging, a significant increase in [Ca2+]e is found in M-type cavities that exclusively support clonal expansion of activated HSCs. This work thus establishes a tool to investigate [Ca2+]e and pH in the HSC niche with high spatial resolution and can be broadly applied to other tissue types.

Extracellular Calcium Ion Concentration Regulates Chondrocyte Elastic Modulus and Adhesion Behavior.

Extracellular calcium ion concentration levels increase in human osteoarthritic (OA) joints and contribute to OA pathogenesis. Given the fact that OA is a mechanical problem, the effect of the extracellular calcium level ([Ca2+]) on the mechanical behavior of primary human OA chondrocytes remains to be elucidated. Here, we measured the elastic modulus and cell–ECM adhesion forces of human primary chondrocytes with atomic force microscopy (AFM) at different extracellular calcium ion concentration ([Ca2+]) levels. With the [Ca2+] level increasing from the normal baseline level, the elastic modulus of chondrocytes showed a trend of an increase and a subsequent decrease at the level of [Ca2+], reaching 2.75 mM. The maximum increment of the elastic modulus of chondrocytes is a 37% increase at the peak point. The maximum unbinding force of cell-ECM adhesion increased by up to 72% at the peak point relative to the baseline level. qPCR and immunofluorescence also indicated that dose-dependent changes in the expression of myosin and integrin β1 due to the elevated [Ca2+] may be responsible for the variations in cell stiffness and cell-ECM adhesion. Scratch assay showed that the chondrocyte migration ability was modulated by cell stiffness and cell-ECM adhesion: as chondrocyte’s elastic modulus and cell-ECM adhesion force increased, the migration speed of chondrocytes decreased. Taken together, our results showed that [Ca2+] could regulate chondrocytes stiffness and cell-ECM adhesion, and consequently, influence cell migration, which is critical in cartilage repair.

Sulfated alginate based complex for sustained calcitonin delivery and enhanced osteogenesis

Direct administration of salmon calcitonin (sCT) by subcutaneous or intramuscular injection is limited by its low efficiency. Drug delivery systems with sustained delivery properties and high bioactivity are urgently needed. For clinical applications an economical and effective carrier is required, which has been a challenge until now. In this study, a simple alginate/alginate sulfate-sCT (Alg/AlgS-sCT) complex was successfully constructed for sustained release of sCT. The negatively charged sulfate groups facilitate bonding with sCT, which avoids the burst release of sCT and extends the release time up to 15 d (it is only 2 d for pure sCT). More importantly, the bioactivity of the released sCT is not affected during such a long release time, suggesting a conformation similar to that of native sCT. In vitro analysis implies that the complex is biocompatible. Moreover, the combination of AlgS and sCT synergistically improves the osteogenic ability of MC3T3 cells, which show higher alkaline phosphatase levels and intracellular and extracellular calcium ion concentrations. The concentration of intracellular calcium ions is 5.26-fold higher than in the control group after 10 d of incubation. This simple yet effective system has potential applications in clinical trials and may inspire the design of other protein delivery systems.

Molecular and Pharmacological Modulation of CALHM1 Promote Neuroprotection against Oxygen and Glucose Deprivation in a Model of Hippocampal Slices.

Calcium homeostasis modulator 1 (CALHM1) is a calcium channel involved in the regulation of cytosolic Ca2+ levels. From a physiological point of view, the open state of CALHM1 depends not only on voltage but also on the extracellular concentration of calcium ([Ca2+]) ions. At low [Ca2+]e or depolarization, the channel is opened, allowing Ca2+ influx; however, high extracellular [Ca2+]e or hyperpolarization promote its resting state. The unique Ca2+ permeation of CALHM1 relates to the molecular events that take place in brain ischemia, such as depolarization and extracellular changes in [Ca2+]e, particularly during the reperfusion phase after the ischemic insult. In this study, we attempted to understand its role in an in vitro model of ischemia, namely oxygen and glucose deprivation, followed by reoxygenation (OGD/Reox). To this end, hippocampal slices from wild-type Calhm1+/+, Calhm1+/−, and Calhm1−/− mice were subjected to OGD/Reox. Our results point out to a neuroprotective effect when CALHM1 is partially or totally absent. Pharmacological manipulation of CALHM1 with CGP37157 reduced cell death in Calhm1+/+ slices but not in that of Calhm1−/− mice after exposure to the OGD/Reox protocol. This ionic protection was also verified by measuring reactive oxygen species production upon OGD/Reox in Calhm1+/+ and Calhm1−/− mice, resulting in a downregulation of ROS production in Calhm1−/− hippocampal slices and increased expression of HIF-1α. Taken together, we can conclude that genetic or pharmacological inhibition of CALHM1 results in a neuroprotective effect against ischemia, due to an attenuation of the neuronal calcium overload and downregulation of oxygen reactive species production.

Terahertz irradiation-induced motility enhancement and intracellular calcium elevation in human sperm in vitro.

To date, there has been limited evidence to reveal the effect of terahertz radiation on sperm. In this study, semen samples were collected from males who had just finished a prepregnancy computer-assisted semen analysis (CASA). The motility, intracellular concentration of free Ca2+ and DNA integrity of sperm with or without terahertz (0.1 to 3 THz) irradiation at 60 µW/cm2 were assessed. We found that terahertz irradiation for more than 5 minutes significantly increased the progressive motility percentage of sperm, and the DNA integrity was not changed. We also found that the effect of terahertz irradiation on spermatozoa was weakened by reducing the concentration of extracellular calcium ions or by blocking calcium channels.

Reducing Extracellular Ca2+ Induces Adenosine Release via Equilibrative Nucleoside Transporters to Provide Negative Feedback Control of Activity in the Hippocampus.

Neural circuit activity increases the release of the purine neuromodulator adenosine into the extracellular space leading to A1 receptor activation and negative feedback via membrane hyperpolarization and inhibition of transmitter release. Adenosine can be released by a number of different mechanisms that include Ca2+ dependent processes such as the exocytosis of ATP. During sustained pathological network activity, ischemia and hypoxia the extracellular concentration of calcium ions (Ca2+) markedly falls, inhibiting exocytosis and potentially reducing adenosine release. However it has been observed that reducing extracellular Ca2+ can induce paradoxical neural activity and can also increase adenosine release. Here we have investigated adenosine signaling and release mechanisms that occur when extracellular Ca2+ is removed. Using electrophysiology and microelectrode biosensor measurements we have found that adenosine is directly released into the extracellular space by the removal of extracellular Ca2+ and controls the induced neural activity via A1 receptor-mediated membrane potential hyperpolarization. Following Ca2+ removal, adenosine is released via equilibrative nucleoside transporters (ENTs), which when blocked leads to hyper-excitation. We propose that sustained action potential firing following Ca2+ removal leads to hydrolysis of ATP and a build-up of intracellular adenosine which then effluxes into the extracellular space via ENTs.

Denervated aortic smooth muscle tension and cellular membrane current in variations of calcium ion concentration in iso-osmotic solution

Objective To study the influence of high calcium ion environment on the membrane current of denervated aortic smooth muscles in iso-osmotic condition, as well as study the myogenic spontaneous vasomotion of the aortic smooth muscles under different pre-load state and intervention of calcium-channel blockers. Methods Smooth muscle samples were taken from the aortic smooth muscle layer of Kunming mice. The samples were fixed at two-ends in relaxed state and infiltrated in Ringer's solution. After stabilization, a glass microelectrode was adsorbed on the smooth muscle membrane to form gigaohm-seal. The current state of the membrane was observed under the physiological osmotic pressure. Then, the calcium ions concentration in the Ringer's solution was increased from 0.9 mol/L to 1.2 mol/L, and the immediate change of membrane current was observed after changing of osmotic pressure. Finally, the membrane potential change in iso-osmotic condition was observed when the pre-load was increased to 1 g, and the spontaneous vasomotion curves of the samples were recorded. Results In the relaxation state, the membrane current of the smooth muscle was increased significantly, i.e. (10.25±1.34) pA vs. (24.91±3.27) pA (P<0.05), when the calcium ion concentration was increased from 0.9 mol/L to 1.2 mol/L in iso-osmotic condition. When the pre-load was increased, the variation amplitude of the membrane current was increased in iso-osmotic condition, i.e. (10.25±1.34) pA to (15.33±4.33) pA (P<0.05) for the lower calcium ion concentration, and (24.91±3.27) pA to (33.31±7.25) pA (P<0.05) for the higher calcium ion concentration. If only increasing the pre-load, the myogenic spontaneous vasomotion could be increased by 175% for the condition of lower calcium ion concentration (0.9 mol/L). When the calcium ion concentration was increased to 1.2 mol/L, the spontaneous vasomotion frequency of the specimen was increased, and the range of the vasomotion could be further increased by 40%. After pretreatment with the calcium-channel blocker (0.5 g/L nitrendipine), the membrane current and myogenic spontaneous vasomotion were significantly decreased in the higher calcium ion environment, which indicated that the membrane potential and spontaneous vasomotion was dominated by calcium ions. Conclusions The increase of extracellular calcium ion concentration not only can improve the excitability of the aorta smooth muscle cells, but also can cause significant spontaneous vasomotion, and can improve the compliance of the smooth muscle tissue in pre-load variation. The calcium-channel blockers can inhibit calcium-dominated transmembrane currents, reduce myogenic spontaneous vasomotion, and enhance the stiffness of smooth muscle, which may have negative effects on windkessel vessel function. Key words: Denervation; Quick stretching; Overloading; Spontaneous vasomotion; Membrane current

Acceleration of Skin Barrier Restoration via Activation of Transient Receptor Potential Vanilloid 3 (TRPV3) by Agrimonia pilosa Leaf

The formation and maintenance of the skin barrier are controlled by the proliferation and differentiation of epidermal keratinocytes, and impaired skin barrier homeostasis resulting from abnormal keratinocyte function causes many skin disorders, including atopic dermatitis and psoriasis. Keratinocyte differentiation is regulated by intra‐ and extracellular calcium ion concentrations. Recently, it was reported that the transient receptor potential vanilloid 3 (TRPV3) ion channel is functionally expressed in human keratinocytes. Activation of TRPV3 increased intracellular calcium signaling, which in turn increased transglutaminase 1, 3 activities as well as the subsequent formation of cornified cell envelopes in the epidermis. It was also reported that activation of TRPV3 promotes epithelial cell proliferation and wound healing in oral epithelia. Thus, TRPV3 activation may be a potential therapeutic target for skin barrier recovery in various dermatological diseases. The leaves of Agrimonia pilosa Ledeb (Rosaceae, AP), which have traditionally been used in East Asia to treat conditions including sore throat, parasitic infection, and eczema, have been reported to possess anti‐inflammatory and anti‐allergic effects in lipopolysaccharide‐ and ovalbumin‐induced mouse models. We performed whole‐cell patch‐clamp and tape stripping tests to determine whether AP extract enhances barrier recovery via TRPV3 activation. We found that APH2O potently activated human TRPV3 and subsequently activated TGase activity in vitro. We also confirmed that topical application of APH2O improved the recovery of skin barrier disruption induced by tape stripping in mice. APH2O may be useful for preventing and treating skin barrier disorders, including inflammatory skin diseases such as atopic dermatitis and psoriasis.Support or Funding InformationThis research was supported by the Convergence of Conventional Medicine and Traditional Koran Medicine R&amp;D program funded by the Ministry of Health &amp; Welfare through the Korean Health Industry Development Institute (SHIDI) (To. JH Nam, HI15C0256).

Colony formation of highly dispersed Microcystis aeruginosa by controlling extracellular polysaccharides and calcium ion concentrations in aquatic solution

This study isolated extracellular polysaccharides (EPS) as a powder material from cyanobacterial blooms and the powdered EPS was used to trigger colony formation of dispersed unicellular M. aeruginosa by controlling EPS concentration in culture medium. The effect of Ca2+ ions on the colony formation of M. aeruginosa was also investigated, then the interaction between EPS and Ca2+ ions on colony formation was discussed. The results showed that the addition of the powdered EPS into the medium did not cause morphological changes of M. aeruginosa, suggesting that EPS alone would not induce the colony formation of M. aeruginosa. On the other hand, a high concentration of calcium ions (1000 mg/l) caused colony formation. When EPS and Ca2+ ions in the culture medium were adjusted to 200 and 1000 mg/l, respectively, the colony density, the average cell number per colony and the particle size of M. aeruginosa showed ca. 1.7–2.0 times greater values than those in the Ca2+ added medium. Calcium ion contributed to the aggregation of M. aeruginosa via crosslinked reaction with negatively charged M. aeruginosa cells, and the addition of EPS possessing negatively charged functional groups such as carboxy groups could enhance the reaction, promoting the crosslinked reaction between EPS and Ca2+ ions.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

The reasons for the nonmonotonic influence of extracellular calcium ion concentrations on the capacity of CD4+ T cells in human peripheral blood to polyclonal activation

Extracellular calcium ion concentrations ([Ca2+]o) nonlinearly affected the capacity of CD4+ T cells in human peripheral blood (HPB) to polyclonal activation in vitro. The dependencies of the tested parameters of CD4+ T cell activation on [Ca2+]o values corresponded to a curve with two maxima and one local minimum in between. One of the maxima was in the area of the HPB lower [Ca2+]o normal level, whereas the other was even lower, which is characteristic for pathological states of various origin. Such a pattern could be explained by some differences in T cell surface receptors. For the confirmation of this hypothesis we compared receptor-dependent (a mixture of monoclonal antibodies (mAbs) to CD3 and CD28 molecules) and receptor-independent (a mixture of phorbol myristate acetate and ionomycin) T cell activation pathways in HPB at varied [Ca2+]o values. The results evidenced a key role of the T cell intracellular signaling system for the differentiation of their sensitivity to calcium ions. Another reason can be related to the different in vitro rates of CD4+ T cell activation and differentiation stages. To check this assumption, we measured the time dependencies of activated CD4+CD69, CD4+(Tyr-P)high, CD4+CD29+ and CD4+(HLA-DR)+ T cell portions as well as their [Ca2+]o dependencies. The [Ca2+]o concentration was shown to influence the activation and differentiation stages of CD4+ T cell activation in vitro to the same degree. Hence, the earliest induction stages play a key role for the unequal sensitivity to calcium ions of CD4+ T cells in vitro. It was additionally supported by the analysis of the relative accumulation rates of activated CD4+CD69+ T cells in the presence of 0.4, 0.8, and 1.2 mM EGTA. A comparative role of CD4+ T memory cells and their naive precursors was studied in a separate series of experiments. First, we performed the phenotypical analysis of CD4+CD69+ T cells within the curve maxima. The first maximum of the CD4+ T cell portion dependence on calcium ion concentrations was formed on average by 85% CD4+CD45R0highCD69+ memory T cells, whereas the remaining 15% was constituted by naive CD4+CD45RAlowCD69+ T cells. The second maximum was nearly completely formed by CD4+CD45R0+CD69+ memory T cells. A portion of naive CD4+CD45RAlowCD69+ T cells rarely exceeded 2%. The content of CD4+CD69 T cells in these maxima was proportional to the donor age and was expressed as a straight line with R2 = 0.9077. Ionomycin-resistant (IR) memory T lymphocytes are the most probable candidates for the role of CD4+ T cells capable of being activated in the conditions of hypocalcemia. For the confirmation of this assumption we isolated mononuclear cells and their IR fraction from donor peripheral blood. Both cell populations were activated with a mAbs mixture at varied EGTA concentrations. In the first maximum only a small portion of the IR fraction of CD4+CD45RA-CD45R0+ memory T cells was activated. It was the second maximum that demonstrated the activation of the majority of CD4+CD45RA-CD45R0+ memory T cells. Hence, the presence of two activation maxima of CD4+ T cell in the conditions of hypocalcemia could be accounted for by the presence in HPB of two CD4+ memory Tcell populations differing in the calcium-dependent systems of intracellular signaling.

The role of the reduction of extracellular calcium ion concentrations in activation of human peripheral blood T cells

Calcium plays a fundamental role in many essential functions of a living organism. It is well known that many infections can reduce extracellular calcium concentrations ([Ca2+]o) (hypocalcaemia) in human blood. The importance of Ca2+ for the T lymphocyte activation has been known for a long time, but the optimal range of [Ca2+]o levels has not been found yet. The main goal of this work was the study of functional activities of CD4+ cells in human peripheral blood (HPB) in vitro in the conditions of hypocalcemia. Polyclonal activation was induced in whole blood of healthy donors by immobilized monoclonal antibodies (mAbs) toward CD3 molecules (epsilon chain of the T cell receptor) and dissolved mAbs toward CD28 (the major molecule of T cell costimulation). Dependencies of T-cell activation parameters on varied [Ca2+]o in HPB in vitro were studied. Production of interleukins-2 (IL-2), IL-4, IL-17A, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) as well as CD4+CD69+, CD4+CD25+, and CD4+(CYTOKINE)+ T cells were used as activation parameters of the T cell polyclonal activation in HPB. EGTA was used as a tool for the reduction of [Ca2+]o levels. The results demonstrated that the EGTA-stimulated binding of extracellular calcium in HPB caused a nonlinear dependence of T cell activation on [Ca2+]o. No EGTA influence on resting T cells was found. The dependence of the number of CD4+CD69+ T cells on [Ca2+]o was represented as a nonlinear curve with two maxima and a local minimum in between. One of the maxima was in the area of the HPB lower [Ca2+]o normal level, whereas the other one was even lower, which is characteristic for pathological states. The T cell-induced production of IL-2, IL-4, IL-17A, IFN-γ, and TNF-α as well as the populations of CD4+CD25+ and CD4+(CYTOKINE)+ T cells were analyzed in HPB in vitro under the same activation conditions. All the dependencies obtained were similar in shape to that of the CD4+CD69+ T cell population versus EGTA concentration in HPB in vitro and was observed as curves with two maxima. The dependencies of this type were described first. It is likely that the presence of two maxima and a local minimum in between for the dependence of HPB T cell activation parameters on [Ca2+]o concentrations is of great physiological importance because hypocalcemia has been associated with many pathological states.

Involvement of Calcium Ions in Regulation of the Kinetics of the Release of Acetylcholine Quanta Forming Multiquantum Postsynaptic Responses

Experiments on frog neuromuscular synapses using the “sequential subtraction” method developed in our laboratory were performed to investigate the quantum composition of endplate currents and the time parameters of the release of acetylcholine quanta forming multiquantum postsynaptic responses in the presence of varying calcium ion levels. These experiments showed that at physiological levels of transmitter release in response to application of single stimuli of the motor nerve, quanta were released asynchronously, as indicated by the nature of fluctuations in the latent periods of single-quantum responses forming the complete quantum signal. Different reactions in terms of the quantum composition of endplate currents and the kinetics of quantum release were seen when the extracellular calcium ion concentration was increased: quantum composition increased exponentially with increases in calcium concentration from 0.4 to 1.8 mM, while the synchronicity of quantum release reached a peak at a calcium concentration of 1.0 mM. These data lead to the conclusion that changes in the synchronicity of transmitter quantum release in response to each nerve impulse in conditions of modified calcium influx into nerve terminals can be regarded as one of the mechanisms mediating synaptic plasticity.

In Vitro Gene Delivery with Ultrasound-Triggered Polymer Microbubbles

In the work described here, gene delivery using polymer microbubbles triggered by ultrasound in vitro was investigated. The effects of pressure amplitude (0–2 MPa), center frequency (1–5 MHz), pulse length (3–12,000 μs), pulse repetition frequency (5–20,000 Hz) and exposure time (0–30 s) on transfection efficiency and cell viability were examined. The effects of radiation force, calcium ion concentration and timing of treatments were also examined. Cells were successfully transfected with pressure amplitudes as low as 250 kPa. Transfection was most efficient at lower frequencies and longer pulse lengths, with a transfection efficiency of 24.2 ± 2.0% achieved using a center frequency of 1 MHz, pressure amplitude of 1 MPa, pulse length of 12,000 μs and pulse repetition frequency of 5 Hz. Gene delivery was also affected by the extracellular calcium ion concentration and the timing of treatments.