Surface responses in cultures fibroblasts elicited by ethylenediaminetetraacetic acid

Cultures of chick heart fibroblasts were perfused with the chelating agent ethylenediaminetetraacetic acid (EDTA). Cellular responses were observed under phase optics and recorded by time-lapse cinemicrography. In interphasic fibroblasts, EDTA induces cellular contraction followed by continuous protrusion and retraction of ectoplasmic blebs ("surface bubbling"), formation of motile vermiform processes, and production of rotatory ectoplasmic swellings. The contraction and surface bubbling closely resemble the metaphase contraction and "anaphase bubbling" normally displayed by cultured fibroblasts. In dividing cells, EDTA does not affect metaphases, but anaphase bubbling appears and persists; telophasic expansion and migration of daughter cells are prevented. Initiation of new mitoses occurs during and after exposure to EDTA. No cellular responses are induced by calcium, magnesium, or ferrous chelates of EDTA. The EDTA elects are completely reversible on removal of the chelating agent, resulting in the restoration of the normal interphasic cell form and the normal expansion and migration of mitotic products. The EDTA effects are interpreted to result from the chelation and removal of divalent cations from the cell surface. Possible relations to surface activities observed in normal mitosis are considered, and an hypothesis is presented regarding the role of the developing spindle in cation transfer.     

Surface Responses in Cultured Fibroblasts Elicited by Ethylenediaminetetraacetic Acid

Cultures of chick heart fibroblasts were perfused with the chelating agent ethylenediaminetetraacetic acid (EDTA). Cellular responses were observed under phase optics and recorded by time-lapse cinemicrography. In interphasic fibroblasts, EDTA induces cellular contraction followed by continuous protrusion and retraction of ectoplasmic blebs ("surface bubbling"), formation of motile vermiform processes, and production of rotatory ectoplasmic swellings. The contraction and surface bubbling closely resemble the metaphase contraction and "anaphase bubbling" normally displayed by cultured fibroblasts. In dividing cells, EDTA does not affect metaphases, but anaphase bubbling appears and persists; telophasic expansion and migration of daughter cells are prevented. Initiation of new mitoses occurs during and after exposure to EDTA. No cellular responses are induced by calcium, magnesium, or ferrous chelates of EDTA. The EDTA elects are completely reversible on removal of the chelating agent, resulting in the restoration of the normal interphasic cell form and the normal expansion and migration of mitotic products. The EDTA effects are interpreted to result from the chelation and removal of divalent cations from the cell surface. Possible relations to surface activities observed in normal mitosis are considered, and an hypothesis is presented regarding the role of the developing spindle in cation transfer.     

Effect of Copper Speciation on Whole-Cell Soluble Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

Chelating agents protect hydrogenase against oxygen inactivation

The effect of chelation on rate of air inactivation of hydrogenase from Clostridium pasteurianum has been investigated. All chelating agents used, whether water-soluble or water-insoluble, afforded protection against oxygen inactivation. EDTA appeared to be the most effective. Thus, in the absence of EDTA, hydrogenase in aqueous solution was nearly totally inactivated after 1 hour incubation in air, whereas 0.5 M EDTA (which did not affect significantly catalytic activity) allowed 41% retention of the initial activity even after 3 days incubation.     

EDTA chelation effects on urinary losses of cadmium, calcium, chromium, cobalt, copper, lead, magnesium, and zinc

The efficacy of a chelating agent in binding a given metal in a biological system depends on the binding constants of the chelator for the particular metals in the system, the concentration of the metals, and the presence and concentrations of other ligands competing for the metals in question. In this study, we make a comparison of the in vitro binding constants for the chelator, ethylenediaminetetraacetic acid, with the quantitative urinary excretion of the metals measured before and after EDTA infusion in 16 patients. There were significant increases in lead, zinc, cadmium, and calcium, and these increases roughly corresponded to the expected relative increases predicted by the EDTA-metal-binding constants as measured in vitro. There were no significant increases in urinary cobalt, chromium, or copper as a result of EDTA infusion. The actual increase in cobalt could be entirely attributed to the cobalt content of the cyanocobalamin that was added to the infusion. Although copper did increase in the post-EDTA specimens, the increase was not statistically significant. In the case of magnesium, there was a net retention of approximately 85% following chelation. These data demonstrate that EDTA chelation therapy results in significantly increased urinary losses of lead, zinc, cadmium, and calcium following EDTA chelation therapy. There were no significant changes in cobalt, chromium, or copper and a retention of magnesium. These effects are likely to have significant effects on nutrient concentrations and interactions and partially explain the clinical improvements seen in patients undergoing EDTA chelation therapy.     

Calcium modulates the lipid dynamics of rat hepatocyte plasma membranes by direct and indirect mechanisms

Calcium ion decreases the motional freedom of lipid molecules in isolated rat hepatocyte plasma membranes and in sonicated dispersions (liposomes) of the membrane lipid. The decrease in lipid fluidity was monitored by estimation of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. At least two processes are involved in the mode of action of the cation. The first is direct, i.e., observed on addition of calcium to the liposomes, relatively rapid, with a half-time of 10-15 at 37 degrees C, proportional to the calcium concentration in the range 0-4 mM, and readily reversed on addition of excess EDTA. The second mechanism is indirected and requires the presence of the membrane proteins. It occurs relatively slowly, with a half-time of 75 min at 37 degrees C, tends to plateau with a calcium half-saturation concentration of approximately 1 mM, is of greater magnitude than the direct effect, and cannot be reversed on chelation of calcium by EDTA. Moreover, the indirect effect is specific for Ca2+ as compared to other divalent cations and it results in changes in the lipid composition. Stimulation of phospholipase A activity is likely but does not account for the change in fluidity. The direct action of calcium is ascribed to binding to the lipid bilayer, whereas the indirect action probably results from modulation of membrane-bound enzymes which can alter the lipid composition. The effects of calcium on the membrane lipid fluidity may underly certain of its regulatory actions on membrane functions.     

Heliotropic leaf movements in common beans controlled by air temperature

Heliotropic leaf movements were examined in common beans (Phaseolus vulgaris cv Blue Lake Bush) under outdoor and laboratory conditions. Heliotropic leaf movements in well-watered plants were partly controlled by temperature, and appeared to be independent of atmospheric humidity and CO₂ concentration. When environmental conditions were held constant in the laboratory, increased air temperature caused bean leaves to orient more obliquely to a light source. Ambient CO₂, intercellular CO₂, and net photosynthesis were not correlated with the temperature-induced changes in heliotropic movements, nor did they significantly affect these movements directly. The effect of air temperature on leaf movements need not be mediated through a change in leaf water potential, transpiration, or leaf conductance. Air temperature modified laminar orientation in light through its effect on tissue temperature in the pulvinal region, not that of the lamina or petiole. However, under darkness the temperature effects on leaf movements were not expressed. Active heliotropic movements in response to air temperature allowed lamina temperature to remain close to the thermal optimum of photosynthesis. This temperature effect underlies a commonly observed pattern of leaf movements under well-watered conditions: a tendency for leaves to face the sun more obliquely on hot days than cool days.     

Role of EDTA in a simple method for determining toxicity using a bacterial indicator organism

A method to determine toxicity using a bacterium as the indicator organism previously developed (Botsford 1998) perceives most divalent cations as being toxic. Mercury is perceived as the most toxic, followed by cadmium, zinc and copper. It was found that adding 2.5 m EDTA to the reaction would relieve the toxicity of the 15 divalent cations tested. This effect does not appear to be simple chelation. One micromolar EDTA eliminated the toxicity of 1.6 m calcium or 0.006 m mercury. Thirty-six chemicals were tested for their toxicity in the presence and absence of 2.5 m EDTA and 25 ppm calcium. Twenty-one were less toxic and two of these, p-aminobenzoic acid and tetrachloroethylene would no longer appear to be toxic according to the assay when these additions were present. Six chemicals had the same toxicity with and without the additions. Nine chemicals were more toxic when the EDTA and calcium were present. This experiment was repeated with six chemicals and ten times the EDTA concentration and ten times the calcium concentration. The toxicity with 10× was compared with the toxicity with 1× the additions. The toxicity of 4 of the six chemicals changed with the higher concentration of EDTA and calcium when the absorbancy values observed in samples with the lower levels were compared with samples with the higher levels. Obviously before EDTA can be added to mitigate the toxicity of divalent cations, it must be determined how much EDTA is required to eliminate the toxicity by the ions present in the sample. Alternatively, if the nature of the contaminating organic chemical is known, it can be determined what the effect of EDTA and the divalent cation present is on the apparent toxicity of the compound.     

The interaction between calcium and the activation of Na+, K+-ATPase by noradrenaline

Summary The interaction of noradrenaline, various cation chelators and calcium on Na⁺, K⁺-ATPase from rat cerebral cortex plasma membranes was studied. It was shown that chelation of inhibitory cations by EGTA, EDTA and dipyridyl activated Na⁺, K⁺-ATPase to the same extent as noradrenaline but at higher concentrations; increasing concentrations of EGTA depressed the activation by noradrenaline; calcium in the form of a calcium-EGTA buffer depressed Na⁺, K⁺-ATPase at physiological concentrations; the inhibition of Na⁺, K⁺-ATPase by calcium is dependent on the magnesium concentration in the assay and the inhibition by calcium was partially reversed by noradrenaline.     

The multiple effects of ethylenediaminetetraacetate in several model lipid peroxidation systems

Experiments were performed which illustrate the various ways EDTA can influence lipid peroxidation. Either detergent-dispersed linoleate, or liposomes made from extracted microsomal phospholipids were utilized as substrates for peroxidation. Peroxidation was accomplished using Fe²⁺ or Fe³⁺. In systems utilizing Fe²⁺, EDTA chelation facilitated Fe²⁺ autoxidation which in turn caused peroxidation of detergent-dispersed linoleate. Peroxidation was not initiated during EDTA-Fe²⁺ autoxidation when the substrate lipids were in a liposomal configuration. Systems utilizing Fe³⁺ required an enzyme (either xanthine oxidase or NADPH-cytochrome P₄₅₀ reductase) to reduce the iron for peroxidative activity. EDTA chelation of Fe³⁺ enhanced the xanthine oxidase and NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of detergent-dispersed linoleate, presumably by facilitating the reduction of Fe³⁺. Catalase and mannitol inhibited both EDTA-Fe²⁺- and EDTA-Fe³⁺-dependent lipid peroxidation. EDTA-Fe³⁺ was not capable of initiating peroxidation of phospholipid liposomes following enzymatic reduction by either enzyme, but ADP-chelated iron effectively initiated liposomal peroxidation in similar systems. With xanthine oxidase-catalyzed peroxidation of liposomes with ADP-Fe³⁺, the inclusion of EDTA-Fe³⁺ caused a modest enhancement of activity. EDTA-Fe³⁺ greatly stimulated NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of liposomes with ADP-Fe³⁺. In contrast, the addition of EDTA, rather than EDTA-Fe³⁺ inhibited the liposomal peroxidation catalyzed by either enzyme with ADP-Fe³⁺ when the EDTA concentration exceeded the concentration of Fe³⁺.     

The possible involvement of gibberellins and calcium in tipburn of Chinese cabbage: Study of intact plants and detached leaves

Spraying Chinese cabbage seedlings [Brassica pekinensis (Lour.) Rupr.] with the growth retardant daminozide (succinic acid-2,2-dimethylhydrazide) reduced tipburn of the mature plants. As the concentration of daminozide increased, the reduction in tipburn damage was correlated with increased calcium content in young susceptible leaves. This effect was much more pronounced in plants that were misted once a day during the head formation period.Incubation of detached Chinese cabbage leaves for 48 h (in the dark) in solutions which contained either EDTA or EGTA caused characteristic lesions at the leaf tips. The extent of the damage was reduced by including CaCl₂ in the solutions. Leaves which were incubated in a solution of EDTA+GA₃ or EGTA+GA₃ were severely affected, with the latter solution being the more harmful. GA₃ alone did not enhance tipburn. CaCl₂ greatly reduced the effect of a complex of chelating agents and GA₃. Leaves derived from daminozide-treated plants which were incubated in EDTA+GA₃ were less affected with tipburn lesions than leaves of control plants treated with the same solutions. When detached leaves were water-stressed for 24 h prior to incubation in these solutions, the severity of tipburn symptoms increased. The possible interactions between GA, calcium chelation and tipburn development are discussed.Contribution no. 1171-E, 1984 series, from the ARO, The Volcani Center Bet Dagan, Israel.     

p53 and p73 display common and distinct requirements for sequence specific binding to DNA

Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73β proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73β than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73β but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73β than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Δ30) demonstrates the same sequence discrimination as does p73β. In vivo, both p53 and p73β exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites.     

Calcium chelators induce bronchoconstriction in the canine lung periphery

We studied the mechanism by which Na2EDTA, a divalent cation chelator, induces bronchoconstriction in the lung periphery of mongrel dogs as a model of nonspecific small airway hyperresponsiveness. Using a wedged bronchoscope technique, we measured collateral system resistance (Rcs) before and after challenges with aerosolized Na2EDTA. An isotonic solution (4% Na2EDTA, 0.28 osmol/kg) increased Rcs 91 +/- 21%. Na2EDTA increased Rcs in a dose-dependent fashion after challenges of increasing concentration (0, 1, 3, and 6%) or duration (15, 30, 60, and 90 s) with 6% Na2EDTA. Atropine (1 mg/kg iv) significantly (P = 0.01) attenuated the response to an aerosol challenge with distilled H2O. Atropine did not significantly (P = 0.35) alter the response to a challenge with 4% Na2EDTA. Challenge with 6% Na2EDTA (0.42 osmol/kg) increased Rcs to a significantly greater (P less than 0.01) extent than did challenge with 6% CaNa2EDTA (0.37 osmol/kg, 250 +/- 55 vs. 29 +/- 11%, respectively). We conclude that Na2EDTA induces bronchoconstriction in the canine lung periphery in a dose-dependent fashion. As suggested by the Na2EDTA-CaNa2EDTA comparison, hyperosmolality of the solution alone cannot explain this phenomenon. The mechanism does not depend on muscarinic activity and appears to involve chelation of calcium.     

Acute prooxidant effects of vitamin C in EDTA chelation therapy and long-term antioxidant benefits of therapy

Chelation therapy is thought to not only remove contaminating metals but also to decrease free radical production. EDTA chelation therapy, containing high doses of vitamin C as an antioxidant, is often used in the treatment of diseases such as diabetes and cardiovascular diseases but the effectiveness of this treatment may be variable and its efficacy has not been demonstrated conclusively. The objective of this work was to determine if the vitamin C added to standard chelation therapy cocktails was prooxidant. We administered a standard EDTA cocktail solution with or without 5 g of sodium ascorbate. One hour following the standard chelation therapy, there were highly significant prooxidant effects on lipids, proteins, and DNA associated with decreased activities of RBC glutathione peroxidase and superoxide dismutase while in the absence of sodium ascorbate, there were no acute signs of oxidative damage. After 16 sessions of standard chelation therapy, the acute prooxidant effects of vitamin C remained, but, even in the absence of nutrient supplements, there were beneficial long-term antioxidant effects of chelation therapy and plasma peroxide levels decreased. In conclusion, multiple sessions of EDTA chelation therapy protect lipids against oxidative damage. However, standard high amounts of vitamin C added to EDTA chelation solutions also display short term prooxidant effects. The added benefits of lower levels of vitamin C in chelation therapy need to be documented.     

Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.     

Effects of Compounds Affecting Calcium Channels on Phytochrome- and Blue Pigment-Mediated Pulvinar Movements of Cassia fasciculata

Verapamil and nifedipine, known as calcium channel blockers, inhibited the phytochrome-mediated movements induced on Cassia fasciculata leaflets by a light-off signal, whereas they had no effect on the `blue' pigment-mediated movements induced by a light-on signal. LaCl<sub>3</sub> inhibited both types of reactions, but the inhibition of light-induced opening needed a 10 times higher concentration than that of dark-induced closure. Bay K 8644, an activator of calcium channels, increased the rate of dark-induced closure, whereas it had no effect on the light-induced opening. These data suggest that calcium ions are not mobilized in the same way in the two types of movements: possibly from external stores in the phytochrome-mediated reaction and from internal stores in the `blue' pigment-mediated reaction.     

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells

OBJECTIVE: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. METHODS: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. RESULTS: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. CONCLUSIONS: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.     

The role of calcium in agonist-stimulated hydrolysis of phosphatidylinositol in mouse pancreas

The effects of Ca²⁺ on agonist-stimulated hydrolysis of myo-[2-³H]inostol-labelled phosphatidylinositol in mouse pancreas in vitro, were studied. The increase in cytosol Ca²⁺ concentration produced by the ionophore A23187 did not stimulate the breakdown of phosphatidylinositol. Cholecystokinin-octapeptide (CCK-8) stimulated the hydrolysis of phosphatidylinositol under conditions in which intracellular calcium stores were depleted. The breakdown of phosphatidylinositol was stimulated by bethanechol and CCK-8 in Ca²⁺-free Krebs solution, and the addition of Ca²⁺ to the medium potentiated the effects of these agonists. Lanthanum significantly reduced bethanechol and CCK-8 stimulated hydrolysis of phosphatidylinositol in Krebs solution, but was without effect in Ca²⁺-free Krebs solution. The results of this study support the proposal that PI hydrolysis does not occur as a result of Ca²⁺ mobilization and may be involved in Ca²⁺ gating in the pancreas.     

Monitoring of membrane-bound divalent cations in plant mitochondria using chlorotetracycline fluorescence

Chlorotetracycline (CTC) shows a strongly enhanced fluorescence upon addition of mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-cation medium. This indicates the presence of membrane-bound divalent cations. The chelation by CTC of the membrane-bound divalent cations does not affect the oxidation of exogenous NADH significantly. The removal of the bound divalent cations using ethyleneglycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA) and EDTA causes an 80% decrease in CTC fluorescence. Titration of CTC fluorescence (a direct measure of bound divalent cations) and 9-aminoacridine fluorescence (a measure of surface potential) with EGTA and EDTA gives similar curves, although CTC fluorescence responds more slowly to the addition of chelators. The same bound divalent cations appear to be monitored by CTC fluorescence or by 9-aminoacridine fluorescence.     

The Effect of Calcium Ionophores on Fragmented Sarcoplasmic Reticulum

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.