Effect of multiplication stimulating activity (MSA) on intracellular cAMP levels and adenylate cyclase activity in chick embryo fibroblasts.

Stimulation of hamster lymph node cells, splenocytes and thymocytes by the mitogen phytohemagglutinin-P (PHA) was found to be greatly enhanced by addition of 1–10 mM LiCl to the cultures. Lithium enhanced stimulation, as determined by [³H]TdR incorporation, only if added within the first 24 h of culture. The enhancing effect of lithium was specific for this monovalent cation since equivalent concentrations of KCl or NaCl did not induce a similar effect on [³H]TdR incorporation. The divalent cations Mg²⁺ (1–10 mM) and Ca²⁺ (1-1.6 mM), also had an enhancing effect on PHA stimulation. However, addition of Li⁺ to cultures enhanced with Mg²⁺ and/or Ca²⁺ led to an additional potentiation of the response to PHA. These results suggest that Li⁺ modifies a unique early event during stimulation of lymphoid cells by this mitogen.     

Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride.

Addition of LiCl (1–25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [³H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [³H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li⁺ since it was not observed when similar concentrations of Na⁺, K⁺, or Mg²⁺ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na⁺, Ca²⁺, Mg²⁺, or Ca²⁺ + Mg²⁺ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KCl was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [³H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.     

Acceleration of Sodium-Calcium Exchange Activity during ATP-induced Calcium Release in Transfected Chinese Hamster Ovary Cells

The P_2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca²⁺]_i due to Ca²⁺ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca²⁺ release was accompanied by a Ca²⁺- dependent regulatory activation of Na⁺/Ca²⁺ exchange activity: Addition of extracellular Ca²⁺ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca²⁺]_i in Na⁺-free media (Li⁺ substitution) compared to Na⁺-containing media; no differences between Na⁺- and Li⁺-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na⁺ and Ca²⁺, the ATP-evoked rise in [Ca²⁺]_i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca²⁺]_i which eventually came to exceed the declining [Ca²⁺]_i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba²⁺ influx, consistent with regulatory activation of the Na⁺/Ca²⁺ exchanger. The acceleration of Ba²⁺ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na⁺ or when the Ca²⁺ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca²⁺]_i under Na⁺-free conditions, but did not inhibit the ATP-evoked stimulation of Ba²⁺ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca²⁺ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca²⁺ release from intracellular stores through regulatory activation by increased [Ca²⁺]_i. In the absence of extracellular Ca²⁺, the stimulation of exchange activity is short-lived and follows the time course of the [Ca²⁺]_i transient; in the presence of extracellular Ca²⁺, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca²⁺ entry.     

Inhibition of purified bovine liver glutathione reductase with some metal ions

Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al³⁺, Ba²⁺, Ca²⁺, Li⁺, Mn²⁺, Mo⁶⁺, Cd²⁺, Ni²⁺, and Zn²⁺ on purified bovine liver GR. In a range of 10?μM–10?mM concentrations, Al³⁺, Ba²⁺, Li⁺, Mn²⁺, and Mo⁶⁺, and Ca²⁺ at 5?μM–1.25?mM, had no effect on bovine liver GR. Cadmium (Cd²⁺), nickel (Ni²⁺), and zinc (Zn²⁺) showed inhibitory effects on this enzyme. The obtained IC₅₀ values of Cd²⁺, Ni²⁺, and Zn²⁺ were 0.08, 0.8, and 1?mM, respectively. Cd²⁺ inhibition was non-competitive with respect to both GSSG (Ki_GSSG 0.221?±?0.02?mM) and NADPH (Ki_NADPH 0.113?±?0.008?mM). Ni²⁺ inhibition was non-competitive with respect to GSSG (Ki_GSSG 0.313?±?0.01?mM) and uncompetitive with respect to NADPH (Ki_NADPH 0.932?±?0.03?mM). The effect of Zn²⁺ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (Ki_GSSG 0.320?±?0.018?mM) and NADPH (Ki_NADPH 0.761?±?0.04?mM), respectively.     

Ability of monovalent cations to replace potassium during stimulation of lymphoid cells

Stimulation of hamster thymocytes, splenocytes, or lymph node cells occurred to a minimal extent in the absence of K⁺. This observation was found for stimulation by T-cell mitogens (phytohemagglutinin and concanavalin A), A B-cell mitogen (lipopolysaccharide), or antigen (KLH). Marginal restoration of the responses to these stimulants occurred in the presence of 0.1 mM K⁺ and responsiveness returned to near maximal levels on addition of 1 mM K⁺ to the cultures. Attempts to restore the responsiveness with other monovalent cations revealed an order of effectiveness of K⁺ ≥ Rb⁺ ? NH₄⁺ ≥ Li⁺. At the 1 mM level K⁺ and Rb⁺ were equally effective in supporting stimulation by phytohemagglutinin while all concentrations of Li⁺ tested (0.1–10 mM) would not support stimulation. However, addition of Li⁺ to cultures reconstituted with 1 mM K⁺ or Rb⁺ revealed that this ion could enhance the phytohemagglutinin response by approximately 100% in the presence of K⁺ and only 30% in the presence of Rb⁺. These data support the hypotheses that the Na,K ATPase must be active for lymphocyte stimulation to occur and that some of the biological effects of Li⁺ on lymphocyte stimulation are mediated at the level of the Na,K ATPase.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

Purification and Characterization of Extracellular Phytase from a Marine Yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis> BG3

The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ (at a concentrations of 5.0 mM), but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline (at concentrations of 1.0 mM and 5.0 mM). The K _m, V _max, and K _cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s^-1, respectively.     

Effects of Heavy Metals on Stomatal Movements in Broad Bean Leaves

How do heavy metals affect stomatal movements and whether water channels are involved in stomatal movements was investigated in broad bean (Vicia faba L.) leaves. Three-week old fully expanded leaves were harvested. Leaf epidermis was peeled off and soaked in the Mes–KOH buffer containing the salts of heavy metals. Stomatal aperture was measured under the microscope. The tested heavy metal ions, such as Hg²⁺, Pb²⁺, Zn²⁺, and La³⁺, partly inhibited stomatal opening in light or closing in darkness at submillimolar concentrations, while K⁺, Na⁺ and Mg²⁺ had no visible effects on stomatal movements. As compared to La³⁺, Hg²⁺ affected stomatal movements more significantly. Stomatal movements were almost completely inhibited under a combined Hg²⁺ and La³⁺ treatment. Apparently, La³⁺, a Ca²⁺ channel blocker, inhibits the changes in the cytosolic Ca²⁺ concentration in guard cells, thus affecting stomatal movements. The inhibitory effect of Hg²⁺ on stomatal movements may be explained by the inhibition of water channels. Like Hg²⁺, Zn²⁺ and Pb²⁺ interfered with stomatal movements. It is concluded that heavy metals at submillimolar concentrations inhibit stomatal movements. They may affect water fluxes through guard cell membranes in different ways, i.e., Hg²⁺, Pb²⁺, and Zn²⁺ inhibit water channels, whereas La³⁺ block ion channels. Water channels may be involved in stomatal movements by regulating water fluxes and play a dominant and primary role in stomatal movements.     

Colorimetric chiral fluorescent sensors for Eu3+ and sequential enantioselective sensing of malate anion

Novel phenanthroline Schiff base fluorescent sensors L1 , L2 , and D1 were designed and synthesized. The sensing abilities of the compounds in the presence of metal cations (Li⁺, Na⁺, K⁺, Ag⁺, Mg²⁺, Ba²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Hg²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Co²⁺, Cu²⁺, Cr³⁺, Fe³⁺, Fe²⁺, Al³⁺, and Eu³⁺) were studied by UV‐vis and fluorescent spectroscopy. The compounds L1 , L2 , and D1 could act as Eu³⁺ ion turn‐off fluorescent sensors based on ligand‐to‐metal binding mechanism in DMSO‐H₂O solution (v/v = 1:1, 10 mM Tris, pH = 7.4). Additionally, the L1 –Eu³⁺ and D1 –Eu³⁺ complexes could be applied as turn‐on enantioselective sensors sensing of malate anion isomers with color changes. Furthermore, biological experiments using living PC‐12 cells demonstrated that L1 and D1 had excellent membrane permeability and could be used as effective fluorescent sensors for detecting Eu³⁺ and malate anion in living cells.     

Effect of divalent metal ions on the microsomal aniline hydroxylase system of rainbow trout

The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25°C. The activity of the aniline hydroxylase system was competitively inhibited by Hg⁺², Ni⁺², Cd⁺², and Zn⁺², while Cu⁺² and Fe⁺³ seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC₅₀ values at fixed aniline concentration were estimated to be 0.45 mM for Hg⁺², Ni⁺², and Cd⁺², 1.8 mM for Zn⁺² and Fe⁺³, and 1.3 mM for Cu⁺². Eadie-Hofstee plots gave identical V_max values of approximately 0.046 nmol/min per mg of protein while K_m values were increased in the presence of Hg⁺², Ni⁺², CD⁺², and Zn⁺², indicating competitive inhibition. Both K_m and V_max values were affected by Fe⁺³ and Cu⁺², suggesting noncompetitive inhibition. K_i values extracted from the Dixon plots were determined t be 0.23, 0.43, and 0.65 mM for Hg⁺², Ni⁺², and Cd⁺², respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The K_i values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions. © 1997 John Wiley & Sons, Inc.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Enhancement of transport in Micrococcus lysodeikticus by sodium ions

Na⁺ (at a concentration of 10 mM) increased the uptake of succinate, glucose and l-valine by Micrococcus lysodeikucus cells considerably. The effect of Na⁺ could be duplicated by Li⁺ only, which, however, was less active. The other cations tested (K⁺, NH₄⁺, Cs⁺, Mg²⁺, Ca²⁺ and Mn²⁺ were ineffective at concentrations up to 100 mM. Addition of Na⁺ increased the affinities of the uptake system for the substrate studied, while uptake capacity remained unaltered.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Na<Superscript>+</Superscript>-mediated piezoprotection in<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis>

Sodium concentrations as low as 2 mM exerted a significant protective effect on the high-pressure inactivation (160–210 MPa) of Rhodotorula rubra at pH 6.5, but not on two other yeasts tested (Shizosaccharomyces pombe and Saccharomyces cerevisiae). A piezoprotective effect of similar magnitude was observed with Li⁺ (2 and 10 mM), and at elevated pH (8.0–9.0), but no effect was seen with K⁺, Ca²⁺, Mg²⁺, Mn²⁺, or NH₄ ⁺. Intracellular Na⁺ levels in cells exposed to low concentrations of Na⁺ or to pH 8.0–9.0 provided evidence for the involvement of a plasma membrane Na⁺/H⁺ antiporter and a correlation between intracellular Na⁺ levels and pressure resistance. The results support the hypothesis that moderate high pressure causes indirect cell death in R. rubra by inducing cytosolic acidification.Communicated by K. Horikoshi     

Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule

Summary The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K⁺ and Ca²⁺ transport and respiration (QO₂) of proximal tubule suspensions. Hg²⁺, Cu²⁺, and Au³⁺ (10^–4 m) each caused a rapid net K⁺ efflux and a delayed inhibition of QO₂. The Hg²⁺-induced net K⁺ release represented passive K⁺ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg²⁺ and Ag⁺ promoted a net Ca²⁺ uptake that was nearly coincident with the onset of the net K⁺ efflux. A delayed inhibition of ouabainsensitive QO₂ and nystatin-stimulated QO₂, indicative of Na⁺, K⁺-ATPase inhibition, was observed after 30 sec of exposure to Hg²⁺. More prolonged treatment (2 min) of the tubules with Hg²⁺ resulted in a 40% reduction in the CCCP-uncoupled QO₂, indicating delayed injury to the mitochondria. The net K⁺ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmaleimide (10^–4 m) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10^–4 m). In addition, both DTT and GSH immediately reversed the Ag⁺-induced net Ca²⁺ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen-deuterium exchange coupled with MALDI-TOF MS

Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A₂ can be activated proteolytically with thrombin and low mM Ca²⁺ or nonproteolytically with high monovalent/divalent cations along with low mM Ca²⁺. Physiologically, FXIII A₂ is poised to respond to transient influxes of Ca²⁺ in a Na⁺ containing environment. A successful strategy to monitor FXIII conformational events is hydrogen–deuterium exchange (HDX) coupled with mass spectrometry. FXIII A₂ was examined in the presence of different cations (Ca²⁺, Mg²⁺, Ba²⁺, Cu²⁺, Na⁺, TMAC⁺, and EDA²⁺) ranging from 1 to 2 mM, physiological Ca²⁺ concentration, to 50–500 mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1 mM Ca²⁺ for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1 mM Ca²⁺ is insufficient for FXIII activation. The metals 1 mM Mg²⁺, 1 mM Ba²⁺, and 1 mM Cu²⁺ each led to conformational changes, many in the same FXIII regions as Ca²⁺. FXIII could also be activated nonproteolytically with 500 mM tetramethylammonium chloride (TMAC⁺) and 500 mM ethylenediamine (EDA²⁺), both with 2 mM Ca²⁺. These different HDX studies help reveal the first FXIII segments that respond to physiological Ca²⁺ levels.     

Effects of heavy metals on the Ca2+-ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.)

1. Heavy metals (Hg²⁺, Cu²⁺, Cd²⁺, Zn²⁺, Pb²⁺) at micromolar concentrations strongly inhibit the Ca²⁺-ATPase activity present in the plasma-membrane obtained from the gill cells of Mytilus galloprovincialis Lam. Heavy metals act through inhibition of the formation of the phosphorylated intermediate.2. All the heavy metals tested inhibit the Ca²⁺-ATPase activity, the effect following the order: Hg²⁺ > Pb²⁺ > Cu²⁺ > Cd²⁺ > Zn²⁺; the simultaneous addition of different heavy metals causes a summatory inhibition of the enzyme activity; addition to the reaction mixture of GSH at a final concentration of 0.5 mM, reverses inhibitory effects of heavy metals.3. The inhibitory effects of Cu²⁺ on Ca²⁺-ATPase are highly enhanced by addition of ascorbate to the reaction mixture. In the presence of ascorbate (100 μM), copper strongly stimulates the lipid peroxidation damage of the gill plasma-membranes, a result that may explain the high copper cytotoxicity.     

Hg2+-induced leakage of electrolytes and inhibition of NO3 − utilization inNostoc calcicola

Summary The effect of mercury (Hg²⁺) in the absence and presence of methylmercury (CH₃Hg⁺), cadmium (Cd²⁺), copper (Cu²⁺), nickel (Ni²⁺) and calcium (Ca²⁺) on Nostoc calcicola Bréb. has been studied in terms of electrolyte leakage, NO₃ ^– uptake and in vivo nitrate reductase (NR) activity to discover any possible correlation among such parameters under Hg²⁺ stress. Leakage of electrolytes from Hg²⁺-treated cyanobacterial cells was directly proportional to Hg²⁺ concentrations and exposure time. In comparison to NO₃ ^– uptake, an about 60-fold slower rate of NR activity was observed in the untreated cultures, the former being five times more Hg²⁺-sensitive. A non-competitive synergistic interaction of Hg²⁺ with CH₃Hg⁺ or Cd²⁺ and antagonistic with that of Ni²⁺ or Ca²⁺ has been observed for both the processes of NO₃ ^– utilization. The antagonistic interaction of Cu²⁺ with Hg²⁺ in terms of NO₃ ^– uptake and synergistic with respect to NR activity, has been attributed to the dual bonding preference of Cu²⁺ for cellular ligands. These findings suggest that (a) a statistically significant correlation exists among such parameters; (b) Hg²⁺ predominantly attacks the cyanobacterial cell membrane; (c) Hg²⁺ inhibits NO₃ ^– utilization; (d) the presence of other cations increases or decreases the inhibitory actions of Hg²⁺.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.