Dinu Georgescauld

Transport of mercury compounds across bimolecular lipid membranes: Effect of lipid composition, pH and chloride concentration

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.

Fundamental roles of biological barriers in mercury accumulation and transfer in freshwater ecosystems (analysis at organism, organ, cell and molecular levels)

In the framework of an ecotoxicological approach to the processes of bioaccumulation and transfer of Hg in freshwater systems, we present a synthesis of our experimental studies concerning the interactions between inorganic Hg and MeHg and biological barriers- at organism and organ levels : three biological models are selected: fish (Salmo gairdneri), burrowing mayfly nymphs (Hexagenia rigida) and rooted macrophytes (Elodea densa, Ludwigia natans). Results show strong specificities of the biological barriers (gills, intestine, roots, ...) towards metal fixation and absorption, closely related to the chemical form of the metal, the contamination sources (water, sediments or food) and the physico-chemical characteristics of the medium ;- at cell and molecular levels : biophysical study of Hg fixation on membrane reveals a new binding site on the phospholipids, the primary amine group on serine and ethanolamine polar heads, jointly with the SH groups of proteins ; Hg(II) induces a strong rigidification of the phospholipidic bilayers. Inorganic Hg and MeHg transports through model membranes (BLM) are essentially due to diffusion of neutral chloride species. These interactions between Hg compounds and membranes are strongly dependent on Hg chemical speciation (pH and pCl effects).

Specific interactions of mercury chloride with membranes and other ligands as revealed by mercury-NMR.

High resolution mercury nuclear magnetic resonance (199Hg-NMR) experiments have been performed in order to monitor mercury chemical speciation when HgCl2 is added to water solutions and follow mercury binding properties towards biomembranes or other ligands. Variations of 199Hg chemical shifts by several hundred ppm depending upon pH and/or pCl changes or upon ligand or membrane addition afforded to determine the thermodynamic parameters which describe the equilibria between the various species in solution. By comparison to an external reference, the decrease in concentration of mercury species in solution allowed to estimate the amount as well as the thermodynamic parameters of unlabile mercury-ligand or mercury-membrane complexes. Hence, some buffer molecules can be classified in a scale of increasing complexing power towards Hg(II): EGTA greater than Tris greater than HEPES. In contrast, MOPS, Borax, phosphates and acetates show little complexation properties for mercury, in our experimental conditions. Evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes has been found. Hg(II) does not form complexes with egg phosphatidylcholine membranes. Interaction with PE and PS model membranes can be described by the presence of two mercury sites, one labile, the other unlabile, in the NMR time scale. In the labile site Hg(PE) and Hg(PS)2 would be formed whereas in the unlabile site Hg(II) would establish bridges between three PE or PS molecules. Calculated thermodynamic data clearly indicate that PE is a better complexing agent than PS. Evidence is also found that complexation with lipids uses at first the HgCl2 species. Interestingly, mercury complexation with ligands or membranes can be completely reversed by addition of decimolar NaCl solutions. Minute mechanisms for mercury complexation with the primary amine of PE or PS membrane head groups are discussed.

Membrane fusion without cytoplasmic fusion (hemi-fusion) in erythrocytes that are subjected to electrical breakdown

There are many reports of hemi-fusion in phospholipid vesicles but few published studies on hemi-fusion in cells. We report evidence from both fluorescence microscopy and freeze-fracture electron microscopy for hemi-fusion in the electrofusion of human erythrocytes. We have also characterised the conditions that favour hemi-fusion as opposed to complete fusion, and discuss the possibility that hemi-fusion might precede complete electrically-induced cell fusion. A membrane probe (DiIC16) and a cytoplasmic probe (6-carboxyfluorescein) were used to investigate the behaviour of doubly-labelled human erythrocytes which were aligned in chains by dielectrophoresis and then exposed to high voltage breakdown pulses. Some of the cells were fused by the pulses, as shown by diffusion of both membrane and cytoplasmic probes from labelled to unlabelled cells. With other cells, the membrane probe diffused into unlabelled cells after the breakdown pulses, without the cytoplasmic probe diffusing into unlabelled cells or leaking into the medium. Membrane fusion (hemi-fusion) thus occurred without cytoplasmic fusion in these erythrocytes. Such cells were irreversibly, but fragilely, attached to their neighbours by the breakdown pulses. There was an inverse relationship between conditions that permit complete fusion and those that favour hemi-fusion, with respect to breakdown pulse length, breakdown voltage and, in particular, osmolarity and temperature. The incidence of hemi-fusion in 250 mM erythritol was twice that in 150 mM erythritol, and hemi-fusion was 5-fold greater at 25 degrees C than at 20 degrees C. Hemi-fused erythrocytes occasionally fused completely on heating to 50 degrees C, demonstrating that hemi-fusion can proceed to complete cell fusion. Freeze fracture electron micrographs of preparations of hemi-fused cells revealed long-lived, complementary depressions and protrusions on the E- and P-fracture faces, respectively, of tightly apposed cells that may mediate hemi-fusion. The possibility that the fusion of closely adjacent human erythrocytes by electrical breakdown pulses may involve an intermediate, shared bilayer structure, which is stable in certain conditions but which can be ruptured by osmotic swelling of the permeabilised cells, is discussed.

Free calcium and calpain I activity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

Transient fluorescence signals from pyrene labeled pike nerves during action potential. Possible implications for membrane fluidity changes.

Abstract Pike olfactory nerves labeled with pyrene and illuminated at 340 nm showed a highly resolved monomer fluorescence emission and a broad excimer emission band at longer wavelength. The excimer formation being controlled by lateral diffusion in the membrane lipids, the ratio of both maxima emission amplitudes is a fluidity parameter and was found to depend on temperature. When these nerves were stimulated, this ratio (F) underwent a small transient decrease ( Δ F F range = 10−3 to 10−4), synchronous with the propagated impulse. These findings may be interpreted as a transient decreased fluidity of the membrane lipids during excitation

FURA-2 IMAGING OF SPONTANEOUS AND ELECTRICALLY INDUCED OSCILLATIONS OF INTRACELLULAR FREE CA2+ IN RAT MYOTUBES

Rat myotubes have a resting [Ca2+]i of about 82 nM. Myotubes 3–5 days old (quiescent myotubes) display electrically induced and spontaneous transients in the intracellular concentration of free Ca2+ ions ([Ca2+]i) uncoupled to any detectable contraction. By contrast, 1-to 2-day-old myotubes are insensitive to electrical stimuli and, after 6 days in culture, stimulated myotubes always show [Ca2+]i transients and twitch contractions. The spatial distribution of [Ca2+]i variations in quiescent myotubes is heterogeneous, local increases in [Ca2+]i being mainly observed near the periphery of the cell. The small effect of different external Ca2+ concentrations and of Cd2+ on the amplitude of the [Ca2+]i oscillation indicates that the main source of Ca2+ may be the sarcoplasmic reticulum. This conclusion is supported by the close similarity between electrically induced and caffeine-induced [Ca2+]i maps. These findings suggest that, at an early stage of myotube ontogenesis, a part of the excitation/contraction coupling, as membrane ionic channels, voltage sensors and Ca2+ release and reuptake mechanisms, is functional but, apparently, still uncoupled to the contractile machinery.

The effects of the insecticide decamethrin on action potential and voltage-clamp currents of Myxicola giant axon

Abstract 1. The effects of the externally applied insecticide decamethrin on nerve excitability was studied on the giant axons of Myxicola infundibulum. 2. Membrane resting potential was only slightly reduced; Spike amplitudes were irreversibly reduced by 8–12%. 3. Voltage-clamp analysis showed a reduction of both early transient and late steady-state conductances. Kinetic effects were a prolongation of the time to peak early transient current and a slowing down of the first component of sodium tail current. 4. These results present similarities with allethrin-treated squid axons. The mechanism of action of pyrethroids is discussed.

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes.

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca2+, 37°C, pH 7.4), 10%–20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca2+]i) of these cells was 106±4nM versus 77±3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity ≈ 50 μm/s, maximal Ca amplitude=195±11nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca2+]i increase induced by K+ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca2+]i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).

Fluorescence quenching study of mercury compounds and liposome interactions: effect of charged lipid and pH.

Abstract In relation to ecotoxicological research on the bioaccumulation and transfer processes of mercury compounds, a study of the interactions between these contaminants and the membrane barriers was investigated. The fluorescence quenching measurement of pyrene (collisional process)—a hydrophobic probe embedded into lipidic dispersions (PC, PS, PC-PS)—was performed with two mercury compounds (HgCl2 and CH3HgCl) versus pH (5.0 and 9.5), lipidic composition of artificial membranes, and chemical speciation. The results show that the accessibility of the mercurials to the hydrophobic core is highly influenced by the parameters studied; these results can be correlated to those observed in mercury bioaccumulation processes in natural environments.