Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Growth analysis of UV-B-irradiated cucumber seedlings as influenced by photosynthetic photon flux source and cultivar

A growth analysis was made of ultraviolet-B (UV-B)-sensitive (Poinsett) and insensitive (Ashley) cultivars of Cucuumis satives L. grown in growth chambers at 600 μmol m⁻² s⁻¹ of photosynthetic photon flux (PPF) provided by red- and far-red-deficient metal halide (MH) or blue- and UV-A-deficient high pressure sodium/deluxe f HPS/DX) lamps. Plants were irradiated 6 h daiiy with 0.2 f-UV-B) or 18.2 C+UV-B) kJ m⁻² day⁻¹ of biologically effective UV-B for 8 or 15 days from time of seeding. In general, plants given supplemental UV-B for 15 days showed lower leaf area ratio (LARs, and higher specific leaf mass (SLM) mean relative growth rate (MRGR) and net assimilation rate (NAR) than that of control plants, but they showed no difference in leaf mass ratio (LMR), Plants grown under HPS/DX lamps vs MH lamps showed higher SLM and NAR. lower LAR and LMR. hut no difference in MRGR. LMR was the only growth parameter affected by cultivar: at 15 days, it was slightly greater in Poinsett than in Ashley. There were no interactive effects of UV-B. PPF source or cultivar on any of the growth parameters determined, indicating that the choice of either HPS/DX or MH lamps should not affect growth response to UV-B radiation. This was true even though leaves of UV-B-irradiated plants grown under HPS/DX lamps have been shown to have greater chlorosis than those grown under MH lamps.     

The binding of zinc,but not cadmium,by phytic acid in roots of crop plants

Plant species adapted to soils enriched with heavy metals often accumulate these metals in their above or below ground organs. In this study, electron probe microanalysis of fractured, quench-frozen root specimens of common crop species shows that an appreciable quantity of Zn can be bound as Zn phytate (myo-inositolkis-hexaphosphate) within small vacuoles of cells in the root elongation zone of lucerne, soybean, lupins, tomato, rapeseed, cabbage, radish, maize and wheat exposed to high levels of Zn (80–300 M). Globular deposits of Zn phytate are most frequently observed in the endodermis of dicotyledonous species and in the pericycle of monocotyledonous species, but may also occur in the stele and inner cortex after prolonged exposure to toxic levels of Zn. The deposits could not be found in Zn-treated sunflower, field peas and Italian ryegrass. In three crop species, lucerne, soybean and maize, Zn-induced phytate globules were frequent, but exposure of roots to 30 M Cd did not induce the formation of Cd-containing globular deposits as observed inLemna minor (Van Steveninck et al., 1990a, 1992). Simultaneous Zn and Cd treatment induced the formation of Zn phytate globules as effectively as Zn alone, and Cd was not detected in the deposits.     

Simultaneous extraction of soil phytoavailable cadmium,copper, and lead by chelating resin membrane

Cadmium, copper, and lead were extracted from suspensions of contaminated soils using metal chelating exchange resin membranes. Nine soils with widely varying properties and Cd, Cu and Pb levels were tested. Soil suspensions made up with 4 g in 40 mL deionized water were equilibrated with 5 cm² Ca-saturated Chelex exchange resin membrane which was retained inside a polypropylene bag and shaken at 150 rpm for 24 hrs. Resin membrane extractable Cd, Cu and Pb of the soils were correlated with Cd, Cu, and Pb uptake by young wheat seedlings grown in these soils and compared with soil Cd, Cu, and Pb extracted by 0.1 M HCl, 0.01 M CaCl₂, and 0.005 M Diethylenetriamine pentaacetic acid (DTPA). The amounts of Cd, Cu and Pb extracted by the Ca-saturated Chelex membrane from all tested soils correlated well with those absorbed by young wheat seedlings. The Ca-saturated Chelex membrane extractable Cd, Cu and Pb of the soil had the strongest correlation with plant uptake Cd, Cu and Pb among the extraction methods we tested. It was demonstrated that the Ca-saturated Chelex membrane extraction is an appropriate method in simultaneously estimating Cd, Cu and Pb phytoavailability of soil and is applicable to a wide range of soils.     

Trace metal interactions in the macroalga Enteromorpha prolifera (O. F. Müller) J.Ag., grown in water of the Scheldt estuary (Belgium & SW Netherlands), in response to cadmium exposure

The littoral macroalga Enteromorpha prolifera was sampled along the Scheldt estuary at Lillo (Belgium), Bath and Ellewoutsdijk (SW Netherlands). The algae were incubated in water from the same locations with salinities of 7, 14 and 23%, at about 50 molquanta m^–2 s^–1 irradiance and 15 °C. One series of cultures was exposed to 400 nm Cd, a second series served as a control. Temporal changes in cadmium, copper, iron, lead and zinc contents of E. prolifera were monitored with atomic absorption spectroscopy in a 21 day experiment. Both synergistic and antagonistic metal interactions became evident in response to cadmium uptake. During the first 8 days of incubation iron contents increased in both cadmium-exposed and control algae. However, iron contents were relatively higher in cadmium-exposed algae. Similar, but weaker synergisms were also found between lead/cadmium and coper/cadmium. Zinc contents decreased in controls and cadmium-exposed algae, but were significantly lower in cadmium-exposed algae. A distinct antagonism between cadmium and zinc was observed in cadmium-exposed algae.     

Presynaptic modulation by eicosanoids in cortical synaptosomes

In continuing experiments to determine the ionic basis of inhibitory presynaptic modulation, rat cortical synaptosomes were employed and receptor-activated K⁺ efflux was determined with a K⁺ sensitive electrode. When synaptosomes were sub-optimally depolarized by veratridine, the addition of agents that activated purinergic, ₂, muscarinic and opioid receptors all promoted K⁺ efflux. With 2-chloroadenosine as a model inhibitory presynaptic modulator, the increased K⁺ efflux evoked by this agent was blocked by the cyclooxygenase inhibitor indomethacin suggesting that arachidonic acid or its metabolites was an intermediary in opening the channel. When arachidonic acid and PGE₂ were tested, both promoted K⁺ efflux that was inhibited by dendrotoxin and mast cell degranulating peptide, two agents that are known to inhibit a delayed rectifier K⁺ current. Our results suggest that via eicosanoid second messengers, inhibitory presynaptic modulators open a sub-class of K channels that hyperpolarize nerve terminals, therefore less Ca²⁺ would enter per nerve impulse and thus the evoked release of neurotransmitters would be decreased.Abbreviations DTX dendrotoxin - MCDP mast cell degranulating peptide - NHGA norhydroguairetic acid - PGE₂ prostaglandin E₂     

Transfer and expression of PCB-degradative genes into heavy metal resistantAlcaligenes eutrophus strains

Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals. The latter might inhibit the biodegradation of the organics and impair bioremediation. Chromosomally located polychlorinated biphenyl (PCB) catabolic genes ofAlcaligenes eutrophus A5,Achromobacter sp. LBS1C1 andAlcaligenes denitrificans JB1 were introduced into the heavy metal resistantAlcaligenes eutrophus strain CH34 and related strains by means of natural conjugation. Mobile elements containing the PCB catabolic genes were transferred fromA. eutrophus A5 andAchromobacter sp. LB51C1 intoA. eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively. The PCB catabolic genes ofA. denitrificans JB1 were transferred intoA. eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation. TheA. eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers. Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals.