Quantitative analysis of the proliferative activity induced in murine thymocytes by concanavalin A.

A quantitative analysis of the proliferative response induced in murine thymocytes by concanavalin A (Con A) is described. Exogenous 3H-thymidine labels 35 to 40% of the newly incorporated TMP residues under optimal conditions. The density label 5-bromo-2-deoxuridine (BrUdR) does not affect DNA metabolism in this system. With this nucleoside, it is shown that newly synthesized DNA is the result of semi-conservative replication, not repair. Double labeling of DNA provides a monitor for cells traversing the cell cycle (S phase to subsequent S phase). The average cycle time is 12.5 hr, and the shortest cell cycle time is 10 hr. The growing fraction of active cells is about two-thirds. The data show that different subpopulations of thymocytes begin proliferating after various times in culture. Once effectively stimulated by Con A, some of the cells can traverse the cell cycle at least twice more after the mitogen is removed.     

Delta 9-tetrahydrocannabinol suppresses concanavalin A induced increase in cytoplasmic free calcium in mouse thymocytes.

It has been shown that delta-9-tetrahydrocannabinol (THC) suppresses thymocyte, lymph node, and splenic lymphocyte proliferation in response to a mitogenic stimulus. It has also been reported that increases occur in the cytosolic free calcium concentration (Ca2+) in mitogen treated lymphocytes. In an attempt to understand a portion of the molecular basis of the THC induced suppression of lymphocyte proliferation, we have examined the effects of THC on the Concanavalin A (Con A) induced cytosolic free Ca2+ mobilization in mouse thymocytes measured by fluorescent Ca2+ probes and spectrofluorometry. The results show that a 10 minute pretreatment with THC suppresses the normal rise in intracellular free Ca2+ in response to Con A. A THC concentration of 4 micrograms/ml (13 microM) was suppressive and the drug vehicle, DMSO, had no effect. In addition, we found that THC pretreatment did not inhibit the binding of FITC labeled Con A to the thymocytes suggesting that the drug did not interfere with lectin binding to the cell surface. To further define the nature of the Ca2+ response affected by THC, mouse thymocytes containing fura-2 were exposed to Con A either in the presence or absence of Ca(2+)-containing medium. It was observed that THC abrogated both intracellular release (measured in Ca(2+)-free medium) as well as extracellular Ca2+ influx. These results suggest that a portion of the proliferation defect in THC treated lymphocytes may be related to a drug induced inhibition of Ca2+ mobilization that normally occurs following mitogen treatment.     

Inhibitors of arachidonic acid metabolism eliminate the increase in cytosolic free calcium induced by the mitogen concanavalin A in rat thymocytes

Using inhibitors of arachidonic acid (AA) metabolism, the possible involvement of AA products in the generation of [Ca2+]i and the pHi rise induced by the mitogen concanavalin A (Con A) in rat thymocytes has been studied. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) and the phospholipase A2 inhibitor bromophenacyl bromide (10 microM) eliminated the [Ca2+]i signal induced by Con A; the cyclooxygenase blocker indomethacin also inhibited it. However, neither NDGA nor indomethacin suppressed the pHi rise stimulated by Con A. Exogenous AA induced an increase in [Ca2+]i but not in the pHi. These results indicate that AA metabolites, probably of the lipoxygenase pathway, take part in the generation of the [Ca2+]i response to the mitogen. In contrast, they appear not to be involved in the pHi rise evoked by Con A.     

Role of calcium and ROS in cell death induced by polyunsaturated fatty acids in murine thymocytes

We investigated the mechanisms whereby omega‐3 and ‐6 polyunsaturated fatty acids (PUFAs) cause cell death of mouse thymocytes using flow cytometry, focusing on the respective roles of intracellular calcium concentration, [Ca²⁺]_i and reactive oxygen species (ROS). We applied the C‐22, 20, and 18 carbon omega‐3 (DHA, EPA, ALA) and omega‐6 (DTA, ARA, and LNA) fatty acids to isolated thymocytes and monitored cell death using the DNA‐binding dye, propidium iodide. When applied at 20 µM concentration, omega‐3 fatty acids killed thymocytes over a period of 1 h with a potency of DHA > EPA > ALA. The omega‐6 PUFAs were more potent. The C18 omega‐6 fatty acid, LNA, was the most potent, followed by DHA and ARA. Cell death was always accompanied by an increase in the levels of [Ca²⁺]_i and ROS. Both increases were in proportion to the potency of the PUFAs in inducing cell death. Removing extracellular calcium did not prevent the elevation in [Ca²⁺]_i nor cell death. However, the intracellular calcium chelator, BAPTA, almost totally reduced both the elevation in [Ca²⁺]_i and cell death, while vitamin E reduced the elevation in ROS and cell death. BAPTA also prevented the elevation in ROS, but vitamin E did not prevent the elevation in [Ca²⁺]_i. Thapsigargin, which depletes endoplasmic reticulum calcium, blocked the elevation in [Ca²⁺]_i, but CCCP, a mitochondrial calcium uptake inhibitor, did not. These results suggest that the six PUFAs we studied kill thymocytes by causing release of calcium from endoplasmic reticulum, which causes release of ROS from mitochondria which leads to cell death. J. Cell. Physiol. 225: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A.

The effect of the mitogenic lectin concanavalin A on the membrane potential of murine lymphocytes was investigated by observing the fluorescence of cells stained with carbocyanine and oxonol dyes. We describe a rapid and reliable method for detecting lectin-induced membrane potential changes in individual cells by flow cytometric analysis of oxonol fluorescence. By 10 min after addition of lectin to suspensions of isolated cells from lymph node, 7-15% of the cells have responded by releasing oxonol dye, indicating a membrane hyperpolarization. The dose onset of this response is similar to that for mitogenesis, which was assessed by measuring [3H]thymidine incorporation. The effect is abolished by alpha-methyl mannoside (100mM), which prevents concanavalin A from binding to the cells, but not by fucose (100mM). When cells are treated with lectin in medium from which Ca2+ has been omitted or to which quinine (0.5mM) has been added, a membrane depolarization is observed. Since these are conditions under which activation of plasma membrane Ca2+-dependent K+ channels is prevented, these findings support the view that the early hyperpolarization of these cells is brought about by an increase in intracellular free [Ca2+].     

Beta-endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes

Lymphocytes stimulated by mitogens or antigens exhibit an enhanced calcium uptake early in the proliferation or activation response. Modulation of this calcium uptake results in alterations of proliferation and immunocompetence. beta-endorphin and other opioids affect several parameters of lymphocyte competence. Limited data are available concerning the mechanism(s) of these effects. This study examines whether a possible opioid mechanism is the modification of the early calcium influx into stimulated lymphocytes. The time course of both concanavalin A (Con A) and phytohemagglutinin (PHA)-stimulated 45Ca2+ uptake into thymocytes was characterized to determine the optimal time for testing the effects of opioids. beta-Endorphin 1-31 significantly enhanced Con A-stimulated 45Ca2+ uptake into rat thymocytes. This peptide had no significant effect on PHA-stimulated 45Ca2+ uptake or on basal thymocyte 45Ca2+ flux. The beta h-endorphin stimulatory effect was titratable in the range of 0.1 nM to 10 microM. Naloxone did not reverse the enhancement. Met-enkephalinamide and other opioid agonists did not duplicate the stimulatory effect. Thus, the beta h-endorphin 1-31 enhancement of Con A-stimulated 45Ca2+ uptake by rat thymocytes does not operate via classical opioid receptor mechanisms. beta h-endorphin 1-31 appears to be acting on a subset of T cells that are responsive to Con A but not to PHA.     

12-0-tetradecanoylphorbol-13-acetate and concanavalin A enhance glucose uptake in thymocytes by different mechanisms

The effects of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) and the plant lectin concanavalin A (Con A) on glucose uptake in murine thymocytes were studied. TPA induces a rapid dose-dependent increase in the uptake of 2-deoxyglucose and in the transport of 3-0-methylglucose. Con A also elicits a time- and dose-dependent enhancement of 2-deoxyglucose uptake. The effect of Con A, however, is less pronounced. The effect of combined treatment of thymocytes with Con A and TPA is not additive. Cytochalasin B completely inhibits the basal, as well as TPA- and Con A-enhanced, 2-deoxyglucose uptake. Dexamethasone markedly inhibits basal 2-deoxyglucose uptake, but is less inhibitory to enhanced 2-deoxyglucose uptake induced by TPA and Con A. The effect of TPA on 2-deoxyglucose uptake and 3-0-methylglucose transport is refractory to inhibition by isobutyl methyl xanthine, dibutyryl cyclic AMP, and ethyleneglycol tetraacetic acid. These agents markedly inhibit the enhancement of 2-deoxyglucose (2-DOG) uptake by Con A. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, also selectively inhibits Con A enhancement of 2-DOG uptake. Taken together, the results suggest that Con A and TPA exert their stimulatory effect on glucose uptake by different activating mechanisms, but they may share a final common transport pathway.     

A novel early effect of concanavalin A on thymocytes.

Concanavalin A, added to freshly isolated rabbit thymocytes, markedly enhanced the extracellular appearance of non-immunoglobulin proteins. Time course studies revealed that the onset of enhancement occurred virtually without delay. The effect appeared to be restricted only to certain of the thymus-derived cells because thymocytes obtained from rabbits treated with hydrocortisone, as well as splenocytes derived from untreated rabbits essentially did not exhibit the enhancement. Stimulation by concanavalin A was specific in that pokeweed mitogen and lipopolysaccharide were without effect and also in that α-methyl-mannoside, but not galactose, abrogated the concanavalin A-mediated enhancement. Experiments with mouse thymocytes demonstrated that the cells which responded to concanavalin A were primarily cells that bear the θ-antigen on their surface (T-cells).     

Periodate induced cross-linking of concanavalin A-reactive membrane proteins of rabbit thymocytes.

Purified plasma membranes of rabbit thymocytes are exposed to sodium periodate and galactose oxidase at conditions similar to those used to induce mitogenic transformation of lymphocytes. The membrane proteins are then fractionated by dodecyl sulfate poly-acrylamide gel electrophoresis. At concentrations of 0.005 M, Na IO4 cross-links 55,000 D and 110,000 D glycoproteins which are known to specifically bind concanavalin A. Galactose oxidase has a similar cross-linking effect, but, at the same time causes proteolytic degradation of membrane proteins. Our data indicate that oxidizing agents, like NaIO4 and galactose oxidase, can indeed cross-link receptors of the thymocyte plasma membrane as has often been proposed as a possible mechanism of their action.     

Phosphoinositide metabolism and the calcium response to concanavalin A in S49 T-lymphoma cells. A comparison with thymocytes.

Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.     

Sugar binding properties of various metal ion induced conformations in concanavalin A.

Concanavalin A is known to undergo a first-order conformational transition when metals are added to the demetallized protein at pH 5.6 (Brown, R.D., III, et al. (1977) Biochemistry 16, 3883--3896). The rate constants for this process, which wer have measured using a polarographic technique, are identical when zinc, cobalt, or manganese occupies S1 and calcium occupies S2. The reducible sugar, p-nitrophenyl alpha-D-mannopyranoside, binds only to the locked conformational structure which is formed upon the addition of metals. The affinity of the protein for sugars is dependent upon occupancy of S1 and S2 and quite sensitive to the identity of the metal in S2. The metals may be removed from the locked protein structure and the protein temporarily retains its ability to bind with sugars but with a considerably lower affinity. The locked form of concanavalin A is unstable at a pH near 2 and unfolds to the unlocked structure with a half-life of 25 min resulting in simultaneous loss of metal and sugar binding.     

Stoichiometry of manganese and calcium ion binding to concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.     

Metabolism of extracellular adenine nucleotides and adenosine uptake by rat thymocytes; the effect of concanavalin A

Thymocytes under cultivation conditions are established to catabolyze rapidly extracellular ATP and AMP which do not penetrate through the plasma membrane. Thymocytes uptake adenosine produced from adenosine nucleotides. Concanavalin A inhibits the extracellular hydrolysis of AMP and adenosine uptake by thymocytes.     

Changes in calcium uptake by liver induced by ferric lactate.

In vitro and in vivo Ca(2+)-uptake by the liver is increased by ferric lactate. In vitro albumin and deferoxamine inhibit ferric lactate effects. Electrophoresis demonstrates the binding of ferric lactate to albumin. In vivo, ferric lactate induces a significant increase of Ca(2+)-uptake by liver, with a maximum of 2.9 nmol/g against 0.66 nmol/g for control livers (P less than 0.005) between 5 and 24 h after administration. This uptake modification is reversible, while the amount of iron (measured as 59Fe taken up) remains constant throughout the experiment. The affinity of ferric lactate for protein and the iron mass-dependence of Ca(2+)-uptake increase support for the hypothesis of a ferric lactate-cell membrane interaction rather than an iron-catalyzed cell injury by lipid peroxidation as the major event leading to an increased Ca(2+)-uptake.     

Calcium ion uptake in isolated pancreas cells induced by secretagogues.

1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein and the Ca2+ ionophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3',5'-monophosphate and dibutyrylic cyclic guanosine 3',5'-monophosphate have no effect on 45Ca uptake. 2. A graphical analysis of the Ca2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca2+ to the membrane and a slow phase representing Ca2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. The 45Ca-exchangeable pool size is increased by both carbamylcholine and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca2+ transfer into the cell most probably through an increase of the cell membrane permeability for Ca2+.     

Activation of lymphocytes by concanavalin A requires calcium ions.

Mouse spleen cells were exposed to a short pulse of the mitogenic lectin concanavalin A (con A). After removal of con A mitogenesis was measured by the incorporation of tritiated thymidine into DNA. It was found: (a) the number of cells responding to con A was proportional to the time of exposure to con A; (b) exposure of cells to con A in the absence of extracellular calcium failed to initiate mitogenesis; (c) for a mitogenic effect an extracellular calcium concentration greater than 10(-5)M was required during the time that the cells were exposed to con A.     

Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5

Using a radioimmunoassay (RIA) for the determination of adenosine 3'5' cyclic monophosphate (cAMP) and an acetylation-RIA procedure to measure guanosine 3'5' cyclic monophosphate (cGMP), we observed that cGMP levels, but not cAMP levels, were significantly elevated in murine thymocytes which had been incubated with preparations containing the thymic hormone, thymosin. Stimulation of intracellular cGMP levels was seen as early as 1 minute after incubation with thymosin fraction 5 and was maximal at approximately 10 minutes. Dose response studies indicated an optimum stimulation of cGMP with a thymosin concentration of 100 microg/ml. A control spleen fraction prepared by an identical procedure as fraction 5 did not affect the levels of either cyclic nucleotide.     

The importance of membrane-bound ribosomes during the activation of thymocytes by concanavalin A.

Concanavalin A (ConA) seleclively enhanced the incorporation of [³H]leucine into a range of proteins of thymocytes incubated in vitro. At the same time ConA seemed to selectively enhance the synthesis of proteins that occurred on membrane-bound ribosomes (extracted from the mitochondrial fraclion with 1% Triton X-100 buffer). The protein synthetic ‘commitment’ of ribosomes was assessed from the stability of ribosomes in 500 mM KC1 before and after puromycin treatment. This indirect method was necessary because of some polysome degradation in the case of membrane-bound ribosomes. Membrane-bound ribosomes were found to be more than 3 times as ‘committed’ as were free ribosomes and ConA increased their commitment by 37–54%. These observations indicate the potential importance of membrane-bound ribosomes in the regulation of thymocyte protein synthesis, particularly during ‘antigenic’ activation, even though this ribosome fraction constituted less than 20% of the total ribosome population.