Influence of Zn2+ and water on the transport properties of a pyrrolidinium dicyanamide ionic liquid.

In order to expand our understanding of a potential zinc-based battery electrolyte, we have characterized the physical and transport properties of the ionic liquid (IL) 1-butyl-1-methylpyrrolidinium dicyanamide ([C4mpyr][dca]) containing various levels of both Zn(2+) and H2O. Detailed measurements of density, viscosity, conductivity, and individual anion and cation diffusion coefficients using pulsed-field-gradient (PFG) NMR combined with NMR chemical shifts and spin-lattice relaxation (T1) NMR experiments provide insights into the motion and chemical environment of all molecular species. We find that the various techniques for probing ion transport and dynamics form a coherent picture as a function of electrolyte composition. Zn(2+) addition causes a moderate reduction in the self-diffusion of the IL anion and cation, whereas the addition of H2O increases ion mobility by increasing the liquid's overall fluidity. Temperature-dependent (13)C T1 experiments of the dca carbon analyzed using Bloembergen-Purcell-Pound fits show monotonic slowing of anion dynamics with Zn(2+) addition, suggesting increased Zn(2+)/dca(-) association. T1 experiments show minimal change in the spin-lattice relaxation of cation or anion upon H2O addition, suggesting that H2O is playing no significant role in Zn(2+) speciation. Finally, we employ a novel electrophoretic NMR technique to directly determine the electrophoretic mobility of the C4mpyr cation, which we discuss in the context of impedance-based conductivity measurements.

The affinity of human erythrocyte porphobilinogen synthase for Zn2+ and Pb2+.

Porphobilinogen synthase activity has been measured in human erythrocyte lysates supplemented with metal-ion buffers to control free Zn2+ and Pb2+ concentrations. The enzyme is activated by Zn2+ with a Km of 1.6 pM and inhibited by Pb2+ with a Ki of 0.07 pM. Pb2+ and Zn2+ appear to compete for a single metal-binding site. The half-time for loss of Zn2+ from the active site, or replacement of Pb2+ by Zn2+, were in the 10-20-min range at 37 degrees C. Zn2+ did not affect the affinity for the substrate 5-aminolevulinate, but Pb2+ reduced it non-competitively. All the experiments were conducted with a blood sample of the common 1-1 phenotype [Astrin, K. H., Bishop, D. F., Wetmur, J. G., Kaul, B., Davidow, B. & Desnick, R. J. (1987) Ann. NY Acad. Sci. 514, 23-29].

Effect of dexamethasone on zinc transport in rat hepatocytes in primary culture.

Incubation of hepatocytes in medium supplemented with 1 microM dexamethasone and 15 or 30 microM ZnCl2 for 24 h resulted in increased intracellular Zn and metallothionein levels. There were a positive correlation between the Zn and metallothionein concentrations. Initial rates of 65Zn influx from balanced salt solutions containing albumin and ZnCl2 to give known free Zn2+ concentrations were measured over 10 min. At 1.6 nM free Zn2+ the influx rate was the same in control and dexamethasone-treated cultures, but at 18 nM free Zn2+ there was an increased rate of influx after dexamethasone treatment. Measurements of 65Zn efflux were quite hard to interpret, because of the existence of at least two intracellular Zn pools, a fast-exchanging and a slow-exchanging pool. The amount of Zn in the fast-exchanging pool appeared to increase as the external free Zn2+ concentration was increased, while the amount in the slow-exchanging pool increased on pre-treatment with Zn and dexamethasone. The fractional efflux of 65Zn from the fast-exchanging pool appeared to increase with the pool size, implying that the rate of Zn efflux increases faster than the concentration of Zn2+ in that pool.

The mechanism of zinc uptake by cultured rat liver cells.

1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain.

Measurement of free Zn2+ ion concentration with the fluorescent probe mag-fura-2 (furaptra).

The fluorescent probe mag-fura-2, previously used to measure [Mg2+], can also be used to measure [Zn2+]. The peak in the excitation spectrum occurs at 323 nm for Zn2+, compared with 335 nm for Ca2+ and Mg2+. This allows simultaneous measurements of [Zn2+] and either [Ca2+] or [Mg2+], by using 3 excitation wavelengths. The dissociation constant for Zn2+ is 20 nM at pH 7.0-7.8, ionic strength 0.15 and 37 degrees C. This allows [Zn2+] to be measured in the range from 0.5 nM to 1 microM. Mag-fura-2 was used to measure [Zn2+] in Zn2+/albumin and Zn2+/histidine mixtures in a physiological buffer at 37 degrees C and pH 7.4. The data obtained enable one to formulate Zn(2+)-buffers for the 1 to 100 nM Zn2+ range.

Lead transport and binding by human erythrocytes in vitro.

Transport and binding of Pb2+ by human erythrocytes were examined for cell Pb contents in the 1-10 microM range, using the 203Pb isotope. Pb2+ crosses the erythrocyte membrane by the anion exchanger, and can also leave erythrocytes by a vanadate-sensitive pathway, identified with the Ca2+ pump. However, Pb2+ exit is very much less than expected from earlier experiments with resealed erythrocyte ghosts [Simons TJB (1988) J Physiol (Lond) 405:105-113] and the distribution of Pb2+ across the erythrocyte membrane is close to equilibrium. The high ratio of erythrocyte to plasma Pb seen in vivo appears to be due to the presence of a labile Pb(2+)-binding component present in erythrocyte cytoplasm.

Lead-calcium interactions in cellular lead toxicity.

The interaction of Pb and Ca with cellular sites depends upon the concentration of free ions present (Pb2+, Ca2+). The ability of Pb2+ to form complexes with simple anions such as Cl- and OH-, the formation of precipitates such as Pb(OH)2 and Pb3(PO4)2, and the ubiquity of Pb as a contaminant in laboratory reagents implies that particular care is needed in order to define the Pb2+ concentration of a solution. The free Pb2+ concentration may be controlled with Pb2+ buffers, and measured with a Pb2+ selective electrode, a fluorescent dye, fura-2, or an NMR indicator, 19F-BAPTA. Pb(2+)-Ca2+ interactions occur in three main situations at the cellular level. Pb2+ and Ca2+ compete at the plasma membrane for transport systems which effect their entry or exit, such as Ca2+ channels, and the Ca2+ pump. Intracellular Ca2+ is buffered to around 10(-7) M by proteins, endoplasmic reticulum and mitochondria. Pb2+ disturbs intracellular Ca2+ homeostasis. Ca(2+)-Pb2+ interactions at mitochondria have been described, but other mechanisms have not yet been explored. Increases in intracellular [Ca2+] act as a signal (or second messenger). Pb2+ interacts with a number of Ca(2+)-dependent effector mechanisms, such as calmodulin (a Ca2+ receptor protein which couples to several enzymes e.g., phosphodiesterase, protein kinases), protein kinase C, Ca(2+)-dependent K+ channels in the plasma membrane and neurotransmitter release. The actions of Pb2+ on neurotransmission may be relevant to Pb(2+)-induced human neuropathy and encephalopathy.

Observations on the chemical nature of lead in human blood serum.

1. The binding of lead to human blood serum, and components of serum, was studied by titration with the addition of Pb(NO3)2 solution, monitoring the free Pb2+ concentration with a Pb2+ electrode, and by equilibrium dialysis. 2. In fresh serum, about 4999 out of 5000 parts of added lead were bound. This suggests that the free Pb2+ concentration is around 1/5000th of the total lead concentration in the serum of normal subjects, i.e. about 1 x 10(-12) mol/l. 3. About 60% of the binding of lead in serum is abolished by standing in air, by dialysis or by treatment with N-ethylmaleimide. This appears to be due to the presence of thiol compounds, mainly cysteine. The remaining 40% appears to be due to protein, mainly albumin.

Intracellular free zinc and zinc buffering in human red blood cells.

Zn2+ has been allowed to equilibrate across the red cell membrane using two agents that increase membrane permeability to this ion: the ionophore A23187 and the specific carrier ethylmaltol. Extracellular free Zn2+ was controlled with EGTA (1,2-di(2-aminoethoxy)ethane-NNN'N'tetra-acetic acid] buffers, except in the case of ethylmaltol, which itself acts as a buffer. Measurement of cellular zinc content at different levels of free Zn2+ facilitated the study of intracellular Zn2+ binding. It was also possible to estimate intracellular free Zn2+ concentration in untreated cells using a "null-point" technique. Intracellular zinc was found to consist of an inexchangeable component of about 129 mumol/10(13) cells and an exchangeable component of 6.7 +/- 1.5 mumol/10(13) cells, with a free concentration of about 2.4 x 10(-11) M. The main component of Zn2+ buffering is hemoglobin, with a dissociation constant of about 2 x 10(-8) M.

Calcium-dependent zinc efflux in human red blood cells.

Zinc efflux from human red blood cells is largely brought about by a saturable mechanism that depends upon extracellular Ca2+ ions. It has a Vmax of about 35 mumol/10(13) cells hr, a Km for external Ca2+ of 1 x 10(-4) M, and a Km for internal Zn2+ of 1 x 10(-9) M. External Zn2+ inhibits with a K0.5 of 3 x 10(-6) M. Sr2+ is a substitute for external Ca2+, but changes in monovalent anions or cations have little effect on the Zn2+ efflux mechanism. It is unaffected by most inhibitors of red cell transport systems, although amiloride and D-600 (methoxyverapamil, a Ca2+ channel blocker) are weakly inhibitory. The transport is capable of bringing about the net efflux of Zn2+, against an electrochemical gradient, provided Ca2+ is present externally. This suggests it may be a Zn2+:Ca2+ exchange, which would be able to catalyze the uphill movement of Zn2+ at the expense of an inward Ca2+ gradient, which is itself maintained by the Ca2+ pump.

Anionic mechanisms of zinc uptake across the human red cell membrane.

1. Zinc is taken up into human red cells by two mechanisms that depend upon the presence of anions. One of these requires bicarbonate ions, is inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and appears to be catalysed by the anion exchanger. The second occurs in the presence of thiocyanate or salicylate ions and may represent transport of a neutral complex with Zn2+. 2. The initial rate of Zn2+ uptake via the anion exchanger is 64 +/- 13 mumol (10(13) cells x h)-1 microM-1 external Zn2+, in the presence of 5 mM-bicarbonate at pH 7.4 and 37 degrees C (+/- S.D.). This is about 1/250 of the corresponding rate of Pb2+ uptake by the anion exchanger. 3. The variation of transport with Zn2+ concentration, HCO3- concentration and pH suggests that the transported species may be ZnCO3Cl- or Zn(HCO3)Cl.OH-. 4. Zinc efflux could not be observed by either of the above routes. This observation suggests that the intracellular free Zn2+ concentration is below 3 nM.

Active transport of lead by the calcium pump in human red cell ghosts.

1. Resealed human red cell ghosts containing lead buffers bring about a net transfer of lead from the cell interior to the outside. This transfer is ATP dependent. 2. The active transport of lead is characterized by a Vmax (maximum velocity) of 11 mmol/(l cells.h) and a KM (Michaelis constant) of 5 x 10(-8) M for internal Pb2+, at pH 6.8 and 37 degrees C. 3. Lead efflux is antagonized by internal calcium, and is inhibited by vanadate with the same IC50 (inhibition constant) with which vanadate inhibits calcium pumping. 4. It is concluded that lead is transported by the calcium pump.

Calcium and neuronal function.

Calcium is unique among metals because its ions have a very large concentration gradient across the plasma membrane of all cells, from 10(-3) M Ca2+ outside, to 10(-7) M Ca2+ inside. This gradient is maintained by the use of metabolic energy through ion pumping, and its existence allows cells to use transient increases in the intracellular Ca2+ concentration as signals, which regulate cell function. In neurones these Ca signals are initiated by electrical activity (action potentials) which open voltage-dependent Ca channels in the plasma membrane, allowing Ca to enter the cell. Intracellular Ca signals can also be produced by transmitters at synapses, which open Ca channels, either directly, or indirectly by causing local depolarization and the opening of voltage-dependent Ca channels. The main effects of Ca signals on neurones are to alter their electrical activity, by modifying the opening and closing of Na and K channels, and to stimulate the release of transmitter substance. Ca has a host of other effects, such as the regulation of metabolic activity, the regulation of cell growth, and the long-term modification of synaptic efficiency, and it is even implicated in the destruction of neurones.

Effects of lead ions on events associated with exocytosis in isolated bovine adrenal medullary cells.

Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1-6 microM) of Pb2+ on stimulus-secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 microM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol-evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor-mediated channels, measured in the presence of tetrodotoxin (2 microM) and ouabain (50 microM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K- and carbachol-evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine-induced channels as well as through voltage-sensitive calcium channels.

Lead enters bovine adrenal medullary cells through calcium channels.

Agents that stimulate secretion also accelerate the rate of Pb uptake into adrenal medullary cells. For example, when cells are suspended in a medium containing 5 microM Pb2+, depolarization by 77 mM K increases the rate of Pb uptake from 12 +/- 1 to 47 +/- 5 mumol/(L cells X min). K-induced Pb uptake has an apparent Km for Pb2+ of 2.6 microM, and is antagonized by Ca2+ with a K0.5 of 1.4 mM. The Ca channel blocker D-600 inhibits Pb entry with a K0.5 of 0.4 microM. Pb uptake is also stimulated by the Ca channel agonist BAY K 8644. These observations suggest that Pb passes through Ca channels. The permeability of the channels to Pb appears to be at least 10 times the permeability to Ca.

The role of anion transport in the passive movement of lead across the human red cell membrane.

Passive Pb transport across the red cell membrane has been studied by measuring Pb uptake from Pb-buffered solutions into resealed ghosts containing EGTA. Over 90% of Pb uptake occurs by a pathway which is inhibited by drugs which block anion transport. The order of effectiveness is 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) greater than phloretin greater than furosemide and bumetanide. Ouabain and cytochalasin B are ineffective. This implicates the anion-exchange mechanism in Pb uptake. The rate of Pb uptake by this route is directly proportional to external Pb2+ and HCO3- concentrations, and inversely proportional to the H+ concentration. These findings suggest that Pb transport depends on the formation of PbCO3 in solution. Pb transport depends upon the presence of a second anion. In the presence of HCO3-, the rate is stimulated in the order ClO4- less than NO3- and CH3CO2- less than F- less than Cl- less than Br- less than I-. The temperature dependence of Pb uptake is similar to that of HCO3-(-)Cl- exchange. Changes in membrane potential appear to influence Pb transport. The effects are small and somewhat variable, but in general a negative internal potential accelerates uptake and reduces exit. A positive internal potential reduces uptake and accelerates exit. These results suggest that Pb is transported on the anion exchanger. Exchange of PbCO3 for a monovalent anion best fits the experimental data, although transport of a ternary PbCO3(-)anion- complex is a possibility.

Passive transport and binding of lead by human red blood cells.

The uptake of Pb into human red blood cells has been studied using Pb buffers. Passive Pb movements can be studied conveniently when the cells are depleted of adenosine 5'-triphosphate (ATP), to eliminate active transport, and of inorganic phosphate, to prevent precipitation of lead phosphate. Pb can cross the membrane passively in either direction. Influx and efflux show similar properties. Passive Pb transport is strongly stimulated by HCO3-, and is reduced by replacing Cl- with ClO4-. It is inhibited by low concentrations of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2.2'-disulphonic acid (DIDS), characteristic inhibitors of anion transport. Pb uptake is unaffected by varying the external concentrations of Na+, K+ and Ca2+. When Pb enters the cell, it binds mainly to haemoglobin. The ratio of bound Pb:free Pb2+ in the cytosol is estimated to be 6000:1. Pb binding to haemoglobin is unaffected by oxygenation. Binding to albumin is quantitatively similar to binding to haemoglobin. The implications of these results for the transport and binding of Pb in the blood are discussed.

Influence of lead ions on cation permeability in human red cell ghosts.

Intracellular Pb2+ ions can replace Ca2+ ions in stimulating the Ca-dependent K permeability of human red blood cells. In metabolically depleted resealed ghosts, the threshold for stimulation of 86Rb efflux by internal Pb2+ is around 5 X 10(-10) M, and stimulation is half-maximal at about 2 X 10(-9) M, and maximal at 10(-8) M Pb2+. There is no effect on 22Na efflux in this concentration range. 86Rb efflux is antagonized by internal Mg2+ ions, and by the channel-blocking drugs quinidine and diS-C2(5), as observed for the Ca-dependent K permeability in red cells. In ghosts containing EDTA, which prevents any internal effects of Pb2+ ions, external Pb2+ increases both 22Na and 86Rb permeability when its concentration exceeds 6 X 10(-7) M. This effect is seemingly unrelated to the Ca-dependent K permeability. This work makes extensive use of Pb2+ ion buffers, and gives information about their preparation and properties.