Replacement of calcium by cadmium ions from Ca-affinity sites localized in different cytoplasmic compartments of Acanthamoeba cells

Summary In Acanthamoeba cells both Ca and Cd may be precipitated in different cytoplasmic compartments forming electron-opaque deposits, as shown in cells treated with glutaraldehyde supplied with either Ca or Cd respectively. It was found by semiquantitative X-ray microanalysis that the transfer of cells containing Cadeposits to glutaraldehyde supplied with Cd causes a considerable replacement of Ca by Cd: in deposits formed at cell membrane, in cytoplasm, and in mitochondria the total weight percentage of Cd amounted to over 90, only in deposits formed in vacuoles the value was about 80. The replacement was not prevented by the presence of Ca in the transfer medium. When cells containing Cd-deposits were transferred to Ca-supplied medium, Cd predominated as well, its total weight percentage also amounting to over 90 in all the examined deposits. The results suggest that calcium bound in different cell structures may be easily replaced by cadmium, but not conversely, which suggests that Cd is more firmly than calcium linked to many cell constituents well preserved by fixation.     

Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells

Summary Carbonic anhydrase (CAH) activity was demonstrated ultracytochemically in the mouse liver cells fixed with 1% glutaraldehyde buffered to pH 7.2 with 0.1 M cacodylate buffer containing 0.1 M sucrose and other aldehyde fixatives. After the fixed 25–40 section were incubated in Hansson's incubation medium containing 0.2 M sucrose, the cobalt phosphate formed by the action of CAH was converted to lead phosphate by immersing the incubated sections into 0.1% lead nitrate aqueous solution.The lead phosphate precipitate was observed very well on the plasma membrane of hepatocytes in Disse space and of endothelial cells or erythrocytes, and very slightly on the external coat of microvilli in bili canaliculi.In the tissues fixed with 4% formaldehyde, the deposits were found very barely on the microvilli in the space of Disse and the plasma membrane of the endothelial cells or the erythrocytes.As the -hydroxyadipaldehyde-fixed tissues showed the highest the CAH activity but had not a good preservation of morphology, this fixative is not suitable for the electron microscopic histochemistry of CAH.The tissue incubated in medium containing Diamox exhibited non-specific deposits in all over the cell, which were lost when the tissue was treated in Diamox solution before incubation.     

DIFFERENCES IN SENSITIVITY TO CA ION LACK BETWEEN MYOBLASTS AND LARGE MYOTUBES FROM CHICKEN BREAST MUSCLE

Large myotubes degenerated in Ca-deficient medium containing Mg ion. Numerous vacuoles appeared in the cytoplasm and then grew larger. The cells were disrupted and eventually detached from the culture dish. The time course of the destruction process differed from cell to cell and the rate of reduction of creatine kinase in the culture dish was constant irrespective of the time after the removal of Ca ion. Most of the myoblasts survived in Ca-deficient medium, after almost all the large myotubes had disappeared. These myoblasts fused to form new myotubes when Ca ion was supplied.
Myotubes formed from myoblasts which had been cultured with Ca-deficient medium for 2 weeks also degenerated on Ca removal.
Sr ion added to Ca-deficient medium did not completely prevent the destruction of myotubes but decreased the rate of their reduction.     

Phytochelatin accumulation and cadmium tolerance in selected tomato cell lines

Four cell lines of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, were selected for their ability to grow in the presence of up to 6 millimolar CdCl₂. The intracellular Cd concentration in these cells was at least 2.3 times higher than in the medium. Growth in media containing higher concentrations of Cd was accompanied by increased production of Cd-binding phytochelatins and a trend toward accumulation of higher molecular weight phytochelatins. At least 90% of the Cd in the most tolerant cells was associated with Cd-phytochelatin complexes. Cell lines maintained an increased tolerance of Cd in the absence of continuous selection pressure.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Effects of chlordecone and malnutrition on immune response in rats

Effect of Chlordecone (Cd) and malnutrition on total body and spleen weights, and plaque forming cells (PFC) were studied. Rats were fed on normal, calcium (CaD), protein (PD) or Ca+P-deficient diets containing 0, 10 or 100 ppm of Cd for 2 or 4 weeks. High (95−100%) mortality was observed in malnourished rats treated with 100 ppm of Cd for 4 weeks. A slight decrease in body weight and an increase in spleen weight was observed in normal but not malnourished rats treated with 10 ppm of Cd for 4 weeks. PFC were significantly increased in both malnourished and Cd-treated rats. Similar increase in PFC was observed in rats fed on CaD but not PD diet containing 10 or 100 ppm of Cd. Whereas, rats fed on Ca+P-D diet containing 100 ppm of Cd exhibited a significant decrease in PFC.     

Ca2+-dependent deposits at the plasmalemma of Chara internodal cells

Electron dense deposits (EDD) were observed on the extracellular side of the plasmalemma of Chara internodal cells by a calcium-glutaraldehyde fixation technique. The number and size of EDD were greatly increased when cells had been preincubated in Ca2+-enriched medium before fixation. The addition of Na+, Mg2+, or La3+ instead of Ca2+ in incubation and fixation media produced no deposits at all, Sr2+ caused deposits with similar distribution to those formed by Ca2+, and Ba2+ addition resulted in deposits localized at different sites within the cell. Microprobe analysis of single EDD from Ca2+ incubated cells ascertained the presence of calcium in these deposits. Possible functions of the Ca2+-binding sites at the plasma membrane of Chara cells are discussed.     

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 μm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes. Received: 26 June 2000 / Accepted: 20 September 2000     

Effects of Ca2+ deficiency, collagenase, and mechanical dispersion on the ultrastructure of cardiac myocytes of adult rats

A new perfusion medium for isolating cardiac myocytes from adult rats was developed, thereby yielding numerous viable cells with few morphological changes. The main factors in the isolation procedure are Ca2+ deficiency, collagenase, and mechanical dispersion. Their effects on the ultrastructure of cardiac myocytes were separately tested. In isolated hearts, perfusion with a medium containing a physiological Ca2+ concentration (2.5 mM, controls) preserved the cellular fine structure well, whereas perfusion with a medium containing 2.5 mM Ca2+ plus 0.05% collagenase caused swelling and disruption of most cells. Perfusion with a Ca2+-deficient medium followed by a medium with a low Ca2+ concentration (25 microM) either containing or lacking collagenase resulted in widening of the T-tubules, reduced electron density of the external lamina and occasional separation, or even dissolution of this layer. Some cells were damaged and hypercontracted. These appeared more numerous in suspensions, that means after mechanical dispersion of the myocardium. However, most of the isolated cells were regularly shaped (up to 30-60 min as shown in another study) and their ultrastructure was only slightly altered. This corresponds to an adequate preservation of the cell membranes proven in earlier membrane transfer studies.     

Effects of salts in promoting the adhesion of amphibian gastrula cells

Presumptive ectodermal and endodermal cells were isolated from the gastrula of the newt Cynops pyrrhogaster, and the effects of salts on embryonic cell adhesion were examined. When the concentrations of NaCl, KCl, MgSO4 and Ca (NO3)2 in the culture medium were increased individually, they all effectively promoted ectodermal cell adhesion. MgSO4 and Ca(NO3)2 promoted endodermal cell adhesion, but NaCl and KCl did not. The culture medium containing increased NaCl (90 mM) nonspecifically promoted ectodermal cell adhesion to the four substrata studied--polystyrene dish, glass, collagen, and fibronectin. The effects of drugs that influence the membrane permeability of Na+ and Ca2+ and the replacement of Na+, Cl-, and Ca2+ with other ions were studied. The results show that the flow of Na+ and Ca2+ into the cytoplasm promotes ectodermal cell adhesion but exerts no influence on endodermal cell adhesion.     

Distribution of cadmium in leaves of Thlaspi caerulescens

Knowledge of the intracellular distribution of Cd in leaves is necessary in order to understand the mechanisms of hyperaccumulation in Thlaspi caerulescens. Ganges and Prayon, two ecotypes accumulating Cd to different levels, were grown in nutrient medium containing varying concentrations (0, 5, 10, 50, and 100 microM) of Cd. Several different approaches were combined in this study to (i) validate the results obtained by a specific method and (ii) establish the link between observations and measurements performed at different scales. In both ecotypes, Cd, localized by autoradiography, was found mainly at the edges of the leaves, but also in points of higher concentration spread over the whole limb surface. This localization was clearly correlated with the necrotic spots observed on Prayon leaves. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation (apoplasm, cell walls, mesophyll protoplasts, and lower epidermis) showed that Cd had similar patterns of distribution in leaf cells of both ecotypes. Cadmium was found both inside the cells and in the cell walls, mainly in the large epidermal cells but also in small epidermal cells. All the methods used agreed well and the results indicated that metal storage in the plants studied involves more than one compartment and that Cd is stored principally in the less metabolically active parts of leaf cells.     

Calcium phosphate deposits in domes of human pancreatic adenocarcinoma (Capan-1) cell cultures

Human pancreatic cells of the Capan-1 line form domes in culture during the stationary growth stage. The domes are thought to be a result of the transport of water and electrolytes by the Capan-1 cells. In older Capan-1 cultures, the epithelial sheets formed thickenings from several layers of cells of which the outermost ones were joined by tight type junctions. In the intracellular space, deposits of insoluble calcium salts were observed. Culture of Capan-1 cells in the presence of fibroblasts prolonged survival of the cultures with intact domes for more than 80 days. The Capan-1 cells proliferated forming multilayers and closed cavities which we called super-domes. X-ray spectrometry and electron diffraction analysis showed that the abundant deposits inside these cavities consisted of calcium phosphate in an apatite structure. The number of these deposits increased with time in culture, and they appeared to be formed at the sites of contact with an extracellular matrix consisting of cell debris. Deposits were not observed within the culture medium. Cells from domes were stained cytochemically for ATPases and alkaline phosphatases and examined by light and electron microscopy. The Capan-1 cells surrounding the domes were differentiated, polarized cells containing placental type alkaline phosphatases on their apical membranes and Ca2(+)-ATPases on their basolateral membranes. These enzymes were thought to play a role in the accumulation of phosphate and Ca2+ ions in the dome cavities, which then formed crystals in the presence of organic compounds produced by lysis of cells of the deepest layers of the super-domes. The crystals of hydroxyapatite observed in standard Capan-1 cell cultures and those cocultured with fibroblasts were assumed to be a result of transepithelial transport of Ca2+ and phosphate ions by these cells.     

Ion-transporting ATPases and matrix mineralization in cultured osteoblastlike cells

Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca2+-ATPase; Ca2+,Mg2+-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na+,K+-ATPase activity was lower in treated than in untreated cells. HCO3--ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix.     

Growth and differentiation of epidermal cells from the rainbow trout established as explants and maintained in various media

Growth and a number of differentiated characteristics of cultured epidermal cells from the rainbow trout Oncorhynchus mykiss were compared using two commercially available serum–free media, a dermal substrate/serum free kit and a serum–containing medium which had been previously optimized for epidermal cell culture. Each medium supported short term growth over 15 days. Only the medium supplied for dermal substrate culture supported longer growth periods. This medium was supplied for use with a collagen/stromal substrate but gave good cultures even without the substrate. Differentiation, measured by examining mucous cells, cytokeratins, epidermal growth factor receptor, gap junction status and ultrastructure showed that serum–free media gave quantitatively and qualitatively superior expression and short term retention of differentiation over serum–containing medium. Epithelial cell growth with expression of differentiated characteristics can be maintained in primary culture in serum–free medium for at least as long as in serum–containing medium. This provides a useful technique for use when serum presence in medium is undesirable or proves toxic to the specialized cell type under investigation.     

Accumulation of cadmium in pancreatic beta cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd2+ and the effects of this ion on secretory activity and metabolism were investigated in beta cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 mumol/kg dry wt. After 60 min of incubation in a Ca2+-deficient medium containing 2.5 microM Cd2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca2+. The incorporation of Cd2+ was stimulated either by raising the concentration of glucose to 20 mM or K+ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K+. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd2+ efflux into a Ca2+-deficient medium. Concentrations of Cd2+ up to 2.5 microM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd2+ concentration was above 10 microM. Basal insulin release was stimulated by 5 microM Cd2+. At a concentration of 160 microM, Cd2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the beta cell uptake of Cd2+ is facilitated by the activation of voltage-dependent Ca2+ channels. Apparently, the accumulation of Cd2+ mimics that of Ca2+ also involving a component of intracellular sequestration promoted by glucose.     

Effects of phosphatidic acid on recombinant protein production by Chinese hamster ovary cells in serum-free culture

When recombinant Chinese hamster ovary (CHO) cells (ATCC CRL-8200) producing human interferon-γ (hIFN-γ) were incubated with exogenously supplied phosphatidic acid (PA) from egg yolk lecithin in a serum-free medium, PA induced increases in both the density of viable cells and concentration of hIFN-γ secreted into the medium. Dispersion of PA with a non-ionic surfactant, Tween 80, further enhanced both cell growth and hIFN-γ production. Replacement of the culture medium containing PA by fresh medium without PA in the course of a static culture did not influence cell growth indicating that PA is required to be continuously present in a serum-free medium to stimulate cell growth. Using a fresh medium containing PA for replacement resulted in significant enhancement of both cell density and hIFN-γ yield. These results suggest that PA is a promising constituent of low-protein serum-free media for the effective production of recombinant proteins.     

ALKALINE EARTH ELEMENTS AND ZOOSPORE RELEASE AND DEVELOPMENT IN PROTOSIPHON BOTRYOIDES

O'Kelley , Joseph C., and Walter R. Herndon . (U. Alabama, University.) Alkaline earth elements and zoospore release and development in Protosiphon botryoides . Amer. Jour. Bot. 48(9): 796–802. Illus. 1961.—Cells of Protosiphon botryoides Klebs from depleted nutrient medium containing Ca were washed and resuspended in fresh complete medium with Ca; or in media with a Sr, Ba or Na replacement, respectively, for Ca; or in an equivalent CaCl₂ solution or deionized water. Zoospore release was observed in these media upon illumination following a 12-hr dark period. Free zoospores were less abundant in Sr-, Ba- and Na-replacement media than in the Ca medium. Zoospore production and release also were depressed in solutions of only CaCl₂ and in deionized water. In the Sr and Ba media, zoospores were formed but not released from the parent cell, as a rule; some zoospores were released in mass within a gelatinous vesicle which did not liquefy and set the zoospores free; these zoospores lost motility and continued development in Sr, producing characteristic, spheroidal clusters of aplanospores. In the Na medium, protoplasmic cleavage preceding zoospore formation was severely inhibited. A study of the reversibility of Sr inhibition of the zoospore-release mechanism revealed evidence of reversion 12 hr after replacement of Sr by Ca. Walls of cells produced in Ca are rich in ruthenium red-positive materials, whereas cells produced under conditions of Sr replacement lack these materials. The significance of these findings in relation to the Ca requirement of other algal species is discussed.     

Cadmium uptake and its effects on growth of tobacco cell suspension cultures

Cadmium uptake and its effects on growth of tobacco cell suspension cultures were examined. Cadmium was shown to accumulate in cells at two or more times the level in the surrounding culture medium. Dry weight accumulation and packed cell volume of the cultures were increased by exposure to 5 ppm Cd in the medium, but exposure to ≥10 ppm Cd resulted in decreased growth. Mitotic indices and total DNA levels indicate that cadmium reduces the rate of cell division at all levels examined.     

Characterization of voltage-sensitive calcium channels in a clonal pituitary cell line

We have pharmacologically characterized voltage sensitive calcium channels (VSCCs) in GH3 cells, an anterior pituitary clonal cell line known to secrete prolactin and growth hormone. Raising the medium K+ concentration from 5 to 50 mM caused an immediate increase in net 45Ca2+ uptake which remained apparent over a 15 minute time course. 45Ca2+ uptake was maximally stimulated nearly 10-fold over basal levels. This K+-induced stimulation of Ca2+ uptake was not prevented by 10-5M tetrodotoxin or by replacing sodium with choline in the assay medium. Ca2+ uptake was, however, inhibited by several VSCC antagonists: nitrendipine, D-600, diltiazem and Cd2+. Further, the novel dihydropyridine VSCC agonists, BAY K8644 and CGP 28392, enhanced 50 mM K+-stimulated 45Ca2+ uptake and these effects were blocked by nitrendipine.     

Effect of calcium on the absorption and translocation of heavy metals in excised barley roots: Multi-compartment transport box experiment

Summary The effect of Ca on the absorption and translocation of Mn, Zn and Cd in excised barley roots was studied using a multi-compartment transport box technique. A radioisotope (⁵⁴Mn,⁶⁵Zn or^115mCd)-labelled test solution was supplied to the apexes of excised roots and the distribution pattern in the roots was examined in the absence or presence of Ca. Results obtained were as follows. Addition of Ca to the test solution reduced the absorption of Mn and inhibited drastically its translocation in excised roots. With increasing concentrations of Ca in test solutions, its inhibitory effects on the absorption and translocation of Mn became severe. Similar results were observed for the absorption and translocation of Zn. Ca in the test solution decreased the absorption and inhibited drastically the translocation of Zn; as in the case of Mn, higher concentrations of Ca had severe effects on these functions. It was also evident that the addition of Ca to the test solution reduced the absorption of Cd at all levels of Cd concentration (1, 10, and 100 μM). Cd absorption decreased with increasing concentrations of Ca in the test solution. However, Ca accelerated the translocation of Cd in excised roots supplied with test solutions containing up to 10μM Cd. At 100μM Cd, addition of Ca caused a negligibly small acceleration of Cd translocation. The accelerating effect of Ca on Cd translocation, especially “xylem exudation”, decreased markedly with the addition of 2,4-dinitrophenol, but not with the addition of chloramphenicol or p-chloromercuribenzene sulphonic acid. When barley plants were supplied with only CaSO₄ during the entire growing period, that is, plants were not supplied with nutrient solution on the last day of this period, Ca had no accelerating effect on Cd translocation in excised roots.