The effect of mitogenic lectins and monoclonal antibodies on intracellular free calcium concentration in human T-lymphocytes.
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ABSTRACT:
SUBMITTER: O'Flynn K
PROVIDER: S-EPMC1153525 | biostudies-other | 1984 Apr
REPOSITORIES: biostudies-other
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Project description:Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.
| S-EPMC1153144 | biostudies-other
Project description:Plant lectins represent a major group of anti-nutritional factors that can be toxic to human and animals. However, the mechanisms by which lectins regulate cell fates are not well-understood. In the present study, the cellular and molecular impacts of three common lectins, agglutinins from wheat germ [wheat germ agglutinin (WGA)], soybean [soybean agglutinin (SBA)], and peanut [peanut agglutinin (PNA)] were examined in zebrafish embryo and liver cells. WGA and SBA were found to induce cell apoptosis both in vitro and in vivo, while PNA stimulated cell proliferation. WGA and SBA reduced levels of B cell lymphoma-2 (Bcl-2), phosphorylation of Bcl-2-associated death promoter (Bad), cyclin-dependent kinase 4 (Cdk4), and phosphorylation of the retinoblastoma (Rb). WGA and SBA also inhibited the activities of cell survival pathways including protein kinase B (Akt), extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), and target of rapamycin (Tor). Furthermore, WGA and SBA shifted the cellular metabolism characterized by reduced expression of glucose-6-phosphate dehydrogenase (g6pd) and increased expression of glutamine synthetase (glul) and glutamate dehydrogenase (glud). However, PNA showed the opposite effects toward these molecular markers compared to those of WGA and SBA. Therefore, our results revealed some plant lectins (WGA and SBA) were toxic while the other (PNA) was mitogenic. Further characterization of the distinct functions of individual lectins should be valuable for both nutrition and other potential applications.
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Project description:In liver cells, the stimulation of alpha 1-adrenoceptors by noradrenaline induces the production of Ins(1,4,5)P3 through the degradation of membrane polyphosphoinositides [PtdIns(4,5)P2]. InsP3 evokes in turn the release of Ca2+ from internal stores. Our results show that the internal perfusion of single guinea-pig hepatocytes with monoclonal anti-PtdInsP2 antibody blocks the rise in cytosolic free Ca2+ concn. ([Ca2+]i) evoked by noradrenaline, an InsP3-dependent agonist, but not by the monohydroxylated bile acid taurolithocholate 3-sulphate, which is known to permeabilize the endoplasmic reticulum. In these conditions, the bile acid elicited either fast or slow fluctuations of [Ca2+]i independently of any InsP3 production. The responses to the bile acid were also observed in the absence of external Ca2+. The presence of intracellular anti-PtdInsP2 antibody does not affect the response to a photolytic release of InsP3 (1.5 microM final concn.) from a caged precursor.
| S-EPMC1132019 | biostudies-other
Project description:Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.
| S-EPMC1544087 | biostudies-literature
Project description:Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent beta-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12, 13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase.
| S-EPMC1220198 | biostudies-other
Project description:Hypo- and hyperthermia affect both primary and secondary hemostasis; however, there are controversial data concerning platelet activation and the underlying mechanisms under hypo- and hyperthermia. The discrepancies in the data could be partly explained by different approaches to hemostatic reactions analysis. We applied a new LaSca-TMF laser particle analyzer for a simultaneous fluorescence and laser scattering analysis of platelet responses at different temperatures. Human platelets were activated by ADP in a wide range of temperatures, and platelet transformations (e.g., a shape change reaction, aggregation and clot formation) and the intracellular calcium concentration ([Ca2+]i) were analyzed by LaSca-TMF and confocal microscopy. The platelet shape change reaction gradually increased with a rising temperature. The platelet aggregation strongly decreased at low ADP concentrations with the augmentation of the temperature and was independent of the temperature at high ADP concentrations. In contrast, the clotting time decreased with a temperature increase. Similar to the aggregation response, a rise in [Ca2+]i triggered by low ADP concentrations was higher under hypothermic conditions and the differences were independent of the temperature at high ADP concentrations. We showed that the key reactions of cellular hemostasis are differentially regulated by temperature and demonstrated for the first time that an accelerated aggregation under hypothermic conditions directly correlated with an increased level in [Ca2+]i in platelets.
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Project description:The objective of this study was to develop an in vitro pharmacodynamic (PD) system to test the impact of protein binding on antiretroviral (ARV) drug effect and intracellular ARV distribution. CD4(+) T cells were isolated from peripheral blood mononuclear cells (PBMCs) and exposed to varying and physiologically relevant concentrations of human serum albumin (HSA) and the ARV drugs efavirenz (EFV), raltegravir (RAL), etravirine (ETR), and enfuvirtide (ENF). The effect of varying extracellular protein concentration on the intracellular distribution of EFV, RAL, and ETR was assessed using ultraperformance liquid chromatography tandem mass spectrometry. HIV infectivity was assessed using an HIV-1 reporter virus expressing an Env-green fluorescent protein (GFP) and quantified using flow cytometry. Increasing extracellular HSA concentration was associated with increased relative infectivity for all drugs tested as well as decreased intracellular concentrations for EFV, RAL, and ETR. Median-effect plots indicate linearity between log10 antiviral effect (fraction of virus affected divided by fraction unaffected) and log10 intracellular drug concentration. The median [interquartile range (IQR)] slope (m) of the median-effect plots was 2.97 (2.26-5.85) for EFV, 3.52 (3.11-3.74) for ETR, and 2.39 (2.15-3.74) for RAL. The intracellular ARV concentrations associated with half-maximal antiviral effect (IC50) of EFV, ETR, and RAL were 1.2 (0.51-5.39), 39.06 (30.10-51.76), and 4.67 (3.91-5.02) ng/ml, respectively. This study demonstrates a significant reduction in cell penetration and antiviral effect of highly bound ARVs due to increasing extracellular concentration of HSA. This study is therefore the first to demonstrate experimentally how protein binding impacts intracellular distribution and the efficacy of ARVs.
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Project description:Contraction of skeletal muscle is triggered by an increase in intracellular calcium concentration that relieves the structural block on actin-binding sites in resting muscle, potentially allowing myosin motors to bind and generate force. However, most myosin motors are not available for actin binding because they are stabilized in folded helical tracks on the surface of myosin-containing thick filaments. High-force contraction depends on the release of the folded motors, which can be triggered by stress in the thick filament backbone, but additional mechanisms may link the activation of the thick filaments to that of the thin filaments or to intracellular calcium concentration. Here, we used x-ray diffraction in combination with temperature-jump activation to determine the steady-state calcium dependence of thick filament structure and myosin motor conformation in near-physiological conditions. We found that x-ray signals associated with the perpendicular motors characteristic of isometric force generation had almost the same calcium sensitivity as force, but x-ray signals associated with perturbations in the folded myosin helix had a much higher calcium sensitivity. Moreover, a new population of myosin motors with a longer axial periodicity became prominent at low levels of calcium activation and may represent an intermediate regulatory state of the myosin motors in the physiological pathway of filament activation.
| S-EPMC10533363 | biostudies-literature
Project description:Microfluorimetric and patch-clamp techniques have been combined to determine the relationship between changes in mitochondrial metabolism, the activity of KATP channels and changes in intracellular free calcium concentration ([Ca2+]i) in isolated pancreatic beta-cells in response to glucose, ketoisocaproic acid (KIC) and the electron donor couple tetramethyl p-phenylenediamine (TMPD) and ascorbate. Exposure of cells to 20 mM glucose raised NAD(P)H autofluorescence after a delay of 28 +/- 1 s (mean +/- S.E.M., n = 30). The mitochondrial inner membrane potential, delta psi m (monitored using rhodamine 123 fluorescence), hyperpolarized with a latency of 49 +/- 6 s (n = 17), and the [Ca2+]i rose after 129 +/- 13 s (n = 5). The amplitudes of the metabolic changes were graded appropriately with glucose concentration over the range 2.5-20 mM. All variables responded to KIC with shorter latencies: NAD(P)H autofluorescence rose after a delay of 20 +/- 3 s (n = 5) and rhodamine 123 changed after 21 +/- 3 s (n = 6). The electron donor couple, TMPD with ascorbate, rapidly hyperpolarized delta psi m and raised [Ca2+]i. When [Ca2+]i was raised by sustained exposure to 20 mM glucose, TMPD had no further effect. TMPD also decreased whole-cell KATP currents and depolarized the cell membrane, measured with the perforated patch configuration. These data are consistent with a central role for mitochondrial oxidative phosphorylation in coupling changes in glucose concentration with the secretion of insulin.
| S-EPMC1134562 | biostudies-other
Project description:1. The effect of rabbit anti-(pigeon erythrocyte) antibodies plus human complement on the concentration of intracellular free Ca2+ in sealed pigeon erythrocyte 'ghosts' was investigated with the photoprotein obelin. 2. The addition of human serum, as a source of complement, to 'ghosts' coated with antibody caused a rapid increase in intracellular free Ca2+ after a lag of 20-40 s, as detected by an increase in obelin luminescence. 3. The increase in obelin luminescence could not be explained by release of obelin into the medium. It was also Ca2+-dependent in that extracellular EGTA abolished the effect and intracellular EGTA inhibited it and required the complete terminal complex (C56789). No effect was seen with C5678. 4. The concentration of intracellular free Ca2+ before addition of complement was approx. 0.3 microM. This increased to a maximum of 5-30 microM after complement addition and then remained constant for at least 1-2 min. 5. Antibody plus complement induced a rapid increase in 42K+ efflux and an inhibition of cyclic AMP formation. 6. When partially purified complement components (C5b-9) were used in 'reactive lysis' it was possible to inhibit the release of macromolecules from pigeon erythrocyte 'ghosts' by extracellular EGTA. 7. It was concluded that the increase in intracellular free Ca2+ concentration caused by anti-cell antibody plus complement occurred before cell lysis and may be involved in the mechanism of complement-induced cell injury.
| S-EPMC1162779 | biostudies-other