Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells

In suspension-cultured cells of Glycine max and Catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. Leakage of a limited pool of electrolytes precedes callose formation, K⁺ representing the major cation lost. Poly-L-ornithine, as well as the ionophores A 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. Digitonin increases the net uptake of Ca²⁺ from the external buffer with a time course parallel to callose synthesis but lagging behind the leakage of K⁺. Nifedipine partly blocks callose synthesis as well as the digitonin-induced increase in net Ca²⁺ uptake. Taken together, the data support the hypothesis that addition of the various substances might indirectly lead to membrane perturbation causing the common event of an increase in net Ca²⁺ uptake which results in callose deposition by a direct activition of the Ca²⁺-dependent and plasma-membane-located 1,3--glucan synthase.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-β-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg²⁺, 1 millimolar Ca²⁺) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex.     

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of β-furfuryl-β-glucoside (FG), an endogenous activator of plant UDP-glucose:(1→3)-β-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized α-furfuryl-β-glucoside also stimulates callose synthase, exhibiting a slightly higher K_a of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using β-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca²⁺, is proposed.     

Effect of calcium on differentiation of Friend leukemia cells

Induction of hemoglobin synthesis of Friend leukemia cells is inhibited by changing the ratio between internal and external Ca²⁺ concentrations. The concentration ratio can be successfully manipulated by the addition of the growth medium of (1) Ca²⁺ channel blocker D600 (90 nM-4 × 10² nM), (2) Ca²⁺ ionophore A23187 (1 × 10²–2 × 10² nM), and (3) EGTA at molar concentrations comparable to the Ca²⁺ concentration of the medium formulation (3 × 10² μM). The observations suggest that a specific ratio between intra- and extracellular Ca²⁺ is required for erythroid differentiation to proceed.     

内钙拮抗剂TMB-8对白皮松花粉管细胞壁构建的影响

应用荧光显微技术、激光共聚焦扫描显微技术、单克隆抗体免疫荧光标记技术以及傅里叶变换显微红外光谱分析(FTIR)等手段,研究了内钙拮抗剂TMB-8对白皮松花粉管胞内Ca2+分布、花粉管生长以及细胞肇构建等的影响.结果表明,白皮松花粉管经TMB-8处理后,胞内的Ca2+浓度下降,花粉管内典型的Ca2+浓度梯度消失,花粉萌发...     

Role of calcium during biosynthesis,secretion and organization of cell-wall polysaccharides

Abstract

The role of calcium during the synthesis, secretion and molecular organization of the primary cell-wall polysaccharides is the topic of this review. With the exception of callose synthase, the in vitro activity of all polysaccharide synthases is not controlled by Ca²⁺ ions. However, changes in the intracellular Ca²⁺ level could control the rate of exocytotic fusion of the secretory vesicles containing cell-wall matrix polysaccharides. In particular, the ability of Ca²⁺ to regulate the fusion of secretory vesicles with the plasma membrane is due to a class of Ca²⁺-dependent phospholipid-binding proteins known as annexins. The ionic interactions between calcium and the negatively charged homogalacturonan domains of the pectins are important not only for the mechanical properties of the wall but also for the gel-properties of these complex biopolymers.     

Direct Photolabeling with [P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase

We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1→3)-β-glucan (callose) synthase of developing fibers of Gossypium hirsutum (cotton). Such a polypeptide migrates coincident with callose synthase during glycerol gradient centrifugation in the presence of EDTA, and can be directly photolabeled with the radioactive substrate, α-[³²P]UDP-glucose. Interaction with the labeled probe requires Ca²⁺, a specific activator of callose synthase which is known to lower the K_m of higher plant callose synthases for the substrate UDP-glucose. Using this probe and several other related ones, several other proteins which interact with UDP-glucose were also identified, but none satisfied all of the above criteria for being components of the callose synthase.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

The effect of membrane stabilizers on phytochrome-controlled anthocyanin biosynthesis in Brassica oleracea

The promotion of anthocyanin synthesis in red-cabbage seedlings by 5 min exposure to R light is inhibited by subsequent application of CaCl₂. The stimulation of dark synthesis of anthocyanin by n-PrOH and by kinetin is also reduced by Ca²⁺ and by cholesterol, both of which are well known to stabilize cell membranes. By contrast, EDTA, which chelates Ca²⁺, promotes dark synthesis of anthocyanin. Assay of native Ca²⁺ extractable from seedlings immersed in EDTA demonstrates that R light exposure promotes a highly significant increase in extractable Ca²⁺. It is suggested that the molecular configuration of the phytochrome molecule affects the ability of a membrane to bind Ca²⁺ and that this in turn affects the permeability to substrates which are required for anthocyanin biosynthesis.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: III. Large bleb formation caused by Sr2+, ionophores X537A and A23187, and compound 4880

Fertilized eggs of Ilyanassa obsoleta form a protuberance which resembles a normal polar lobe when injected with Sr²⁺ or Ca²⁺ by microiontophoresis. Eggs also form a lobe-like protuberance when exposed to any of three drugs: compound $\text{48}\text{80}$, ionophore X537A, and ionophore A23187. Protuberances form more quickly and at lower drug concentrations if additional exogenous Ca²⁺ is added, whereas higher concentrations of Mg²⁺ do not have such an effect. When eggs are exposed to these drugs in Ca²⁺-, Mg²⁺-“free” seawater, with or without 10 mM EDTA, the eggs are still able to undergo extensive shape changes and form protuberances. Drug-induced shape changes are prevented by cytochalasin B, but will still occur in the presence of colchicine. Approximately 75% of Ilyanassa eggs are capable of forming and resorbing their third polar lobe and undergoing cytokinesis in Ca²⁺-, Mg²⁺-“free” artificial seawater (even containing 10 mM EDTA), solutions which by atomic absorption spectroscopy are shown to contain low concentrations of Ca²⁺ (3–5 μM) and Mg²⁺ (1.0–3.5 μM). The data suggest that if Ca²⁺ is required for normal polar lobe formation and cytokinesis, it is derived from intracellular sources or is required in only very low exogenous concentrations (i.e., less than 10^?2 μM free Ca²⁺, in the presence of 10 mM EDTA).     

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca²⁺. At a constant benzyladenine (BA) level (4.4×10^?5 M) 10^?4 to 10^?2 M Ca²⁺ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10^?6 M) concentration of external Ca²⁺, Chl synthesis was relatively more Ca²⁺-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca²⁺ from external sources to cytosol, and/or (b) interacted with Ca²⁺ directly in the pathway of Chl biosynthesis. The need of external Ca²⁺ was evidenced in experiments with modulators of Ca²⁺-transport systems. The reduction of the inward current of Ca²⁺ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N′N′-tetraacetic acid, EGTA) and Ca²⁺-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca²⁺. Inhibitory action of Ca²⁺-channel blockers depended on their kind and concentration: at the 10^?5 M concentration La³⁺>verapamil>nifedipine inhibited Chl formation. In the presence of Ca²⁺, the Ca²⁺-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.     

Ca requirement for aerobic nitrogen fixation by heterocystous blue-green algae

The requirement of Ca²⁺ for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N₂ under microaerobic conditions, Ca²⁺ was not required for growth, at least in concentrations greater than traces. In contrast, Ca²⁺ was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca²⁺ to an aerobic culture without Ca²⁺ promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca²⁺ promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca²⁺ or low oxygen tension was dependent on de novo protein synthesis. The role of Ca²⁺ seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.     

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics,Disrupts Actin Filaments and Affects Cell Wall Architecture

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca²⁺ influx, thereby elevating the cytosolic Ca²⁺ concentration [Ca²⁺]c and disrupting the [Ca²⁺]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca²⁺]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 μg/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺. Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca²⁺ and Na polygalacturonate.     

Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin

1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin.2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca²⁺ for aequorin and at least 3Ca²⁺ for obelin.3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units.4. Other monovalent cations, such as Na⁺ are able to compete with Ca²⁺ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions.5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca²⁺ solutions or of calcium/EGTA buffers.     

Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves

The effect of Ca²⁺ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca²⁺ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca²⁺ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN^? was suppressed by 2 mM Ca²⁺ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN^? or CN^? + 0.1 mM Ca²⁺ but had no influence on respiration and photosynthetic O₂ evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca²⁺ in epidermal peels from pea leaves.     

Heavy metal effects on cytosolic free Ca2+ level in the marine protozoan Euplotes crassus evaluated by confocal laser scanning microscopy

The Ca²⁺ concentration in the cytosol of the marine ciliate protozoan Euplotes crassus has been evaluated utilizing the dye Fluo3-AM and measuring the fluorescent response by confocal laser scanning microscopy (CLSM). Serial sections were piled-up in a three dimensional image of the cell to estimate the cation intracellular spatial distribution and subsequently processed in order to quantify the free Ca²⁺ content.The results clearly demonstrated that nano-to micromolar concentrations of Hg²⁺ added to seawater culture medium caused up to a 12-fold increase in the free cytosolic Ca²⁺ of Euplotes crassus. Exposure of the protozoa to Cu²⁺, an essential element, also caused a sustained increase in the free cytosolic Ca²⁺.Pretreatment of Euplotes crassus with W5, a voltage-dependent Ca²⁺-channel blocker, greatly reduced the deleterious effects of the heavy metals on Ca²⁺ concentration. These results indicate that the increase of free Ca²⁺ concentration in the cytosol of the protozoa, induced by micromolar heavy metal concentrations, could depend on an increased influx of extracellular Ca²⁺ not physiologically compensated by the activity of the different mechanisms involved in Ca²⁺-homeostasis.