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OH− Exchange Induced by Cuprous Ions
Toxicology and Applied Pharmacology 159, 204 –213 (1999) Article ID taap.1999.8736, available online at http://www.idealibrary.com on
Copper Effects on Ion Transport across Lamprey Erythrocyte Membrane: Cl 2/OH 2 Exchange Induced by Cuprous Ions Anna Y. Bogdanova,* Leila V. Virkki,† Gennadii P. Gusev,‡ and Mikko Nikinmaa* *Department of Biology, University of Turku, FIN-20014 Turku, Finland; †Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520; and ‡Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Received January 28, 1999; accepted June 17, 1999
Copper Effects on Ion Transport across Lamprey Erythrocyte Membrane: Cl 2/OH 2 Exchange Induced by Cuprous Ions. Bogdanova, A. Y., Virkki, L. V., Gusev, G. P., and Nikinmaa, M. (1999). Toxicol. Appl. Pharmacol. 159, 204 –213. We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO 4 concentration of 100 mM caused anion-dependent intracellular acidification and increase in Cl 2 influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lagphase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na 1 fluxes in both directions and a net Na 1 uptake. Copper-induced Na 1 transport was partially amiloride(MIA)-sensitive representing Na 1/H 1 exchange. The nature of the amiloride-insensitive fraction of copper-activated Na 1 influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K 1 and Cl 2 volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na 1/H 1 exchange and consecutive volume changes cause the RVD response. © 1999 Academic Press
Key Words: copper; intracellular pH; chloride-hydroxyl exchange.
Although copper is an essential trace element for all living organisms, its ionic forms are highly toxic (Harris and Giltin, 1996; Heath, 1997). Since Cu(II) is the dominant form of the element in oxygen-equilibrated aqueous solutions, most of the studies on the copper toxicology have concentrated on the effects of the cupric ions both on the organismic and cellular level. However, within the organism the partial pressure of oxygen is much lower than in the ambient atmosphere, and copper exists mostly as metal–protein complexes. The latter 0041-008X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
conditions may favor formation and stabilization of cuprous ions. Thus, it remains uncertain as to which copper species exerts the toxic effects in the organism. Being the first contact site between the metal ions and the cell, cell membranes may be the main target of copper action. In fishes exposed to sublethal copper concentrations net loss of ions from the organism results from a disturbance in the function of gill epithelial membranes (Wilson and Taylor, 1993; Hansen et al., 1996). In humans, copper-induced haemolytic anemia results from the destruction of erythrocyte membranes (Fairbanks, 1967; Piriou et al., 1987). Both cupric and cuprous ions are reported to affect membrane function. Divalent copper inhibits Na 1/K 1 ATPase in several different cell types including erythrocytes (Li et al., 1996) hence causing a slow dissipation of transmembrane K 1 and Na 1 gradients. Copper (II) ions also induce an increase in sodium conductivity in human erythrocytes (Gwozdzinski, 1991) and molluscan neurons (Weinreich and Wonderlich, 1987). This speeds up the copper-induced depolarization. Furthermore, cupric ions stimulate Na 1/H 1 exchanger in Ehrlich ascites tumor cells (Kramhoft et al., 1988). In asolectin as well as in renal brush border vesicles treated with micromolar concentrations of cuprous sulphate (stabilized by addition of ascorbate as reducing agent), Karniski (1992) observed a rapid copper-mediated electroneutral Cl 2/OH 2 exchange driven by transmembrane pH gradient. He suggested that cuprous ions act similarly to organometal compounds such as tributyltin incorporating into the lipid bilayer in the form of uncharged pairs with halides (Selwin et al., 1970; Wieth and Tosteson, 1979). At present, it is not known how all the above mechanisms of copper action on cellular ion transport are interrelated. The effects of copper ions on membrane transport can be conveniently studied using lamprey erythrocytes. These cells lack rapid band 3-mediated anion exchange (Onishi and Asai, 1985; Nikinmaa and Railo, 1987; Tufts and Boutilier, 1989; Bogdanova et al., 1998), whereby the copper-induced changes in chloride permeability can easily be monitored. The lack of the anion exchange, furthermore, makes it possible to study primarily the effects of extracellular copper, since Cu 21 uptake in, e.g., human erythrocytes, proceeds mainly via the anion
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exchanger (Alda and Garay, 1990). Lamprey erythrocytes have functional Na 1/K 1-ATPase and amiloride-sensitive Na 1/H 1 exchanger (Nikinmaa et al., 1986; Gusev et al., 1992; Virkki and Nikinmaa, 1994) thus enabling the study of all the reported major targets of copper action in an integrative fashion. We have used lamprey erythrocytes to study (1) the effects of copper ions on the transport of chloride, acid equivalents, sodium and potassium, and consecutive changes in the cell volume using radioactive tracers and patch-clamp technique, (2) the interrelationships between the changes in ion fluxes using ion substitutions and specific inhibitors, and (3) the role played by univalent and divalent copper ions on the membrane level studying copper effects in the presence of sulfhydryl reagents, reducing agents, and varying anion composition of the incubation medium. MATERIALS AND METHODS Chemicals. Stock solutions of CuSO 4 and BaCl 2 were prepared in distilled water. Amiloride (Sigma, St. Louis, USA), 5-(N-methyl N-isobuthyl) amiloride, (MIA, Research Biochemicals International, Natick, USA), and R(1)dihydroindenyloxyalkanoic acid (R(1)-DIOA, Research Biochemicals International, Natick, USA) were dissolved in dimethyl sulfoxide (DMSO), and appropriate amounts of solvent were always added to the controls. N-phenyl maleimide (NPM) is from Sigma. 5,5-Dimethyl-[2- 14C]-oxazolidene-2,4-dione (DMO), 22Na, 36Cl, and 86Rb were obtained from Amersham International (Buckinghamshire, England) or Isotope (St. Petersburg, Russia) and dissolved in distilled water at known specific activities. Ascorbate-containing solutions were prepared fresh before use. Animal handling and experimental procedures. River lampreys (Lampetra fluviatilis, 25–100 g) were maintained in laboratory conditions at 5°C for a minimum of 1 week before their red cells were used for experiments. The lampreys were anaesthetized with n-ethyl amino benzoic acid ethyl ester (1 g/L), and blood samples were drawn from the caudal vessel into heparinized syringes. Cells were then pelleted, washed with a solution containing 154 mM NaCl and 1 mM KCl, and finally resuspended in standard incubation medium containing (in mM) 144 NaCl, 1 KCl, 10 Tris–HCl, 10 glucose (pH 7.4 at 20°C). All the experiments were carried out at 20°C. Final haematocrit of the cell suspension was 3–5% if not stated otherwise. In a preliminary set of experiments the highest sublytic copper concentration was determined by measuring the extracellular haemoglobin concentration after separating the incubation medium and cells by centrifugation. Treatment of lamprey erythrocytes with 100 mM CuSO 4 caused hemolysis which started after 20 –30 min of exposure. At higher copper concentrations extensive hemolysis was observed in less than 20 min of treatment. Thus, the highest concentration of copper used in the experiments was 100 mM. Since even at this concentration, hemolysis was observed during prolonged incubations, it was critical for the interpretation of our results that the integrity of the cell membrane was not disrupted during the time course of the experiments described. For this reason, we excluded all the incubations in which significant hemolysis occurred. The lack of hemolysis indicates that no macromolecular leakage from the cells occurs. Further, we measured unidirectional rubidium fluxes (as a tracer for potassium) across the cell membrane. An increase in potassium permeability is often taken to indicate disturbances in membrane integrity (e.g., Rabergh et al., 1992). In the present experiments, 100 mM copper concentration did not affect the rubidium permeability (Table 2). Together, the lack of copper effects on rubidium permeability and on macromolecular leakage indicate that the membrane integrity was not compromised. Measurements of Na 1, K 1, and Cl 2 uptake and unidirectional fluxes. The measurements were carried out either in the absence or presence of transport inhibitors, in the absence or presence of sulfhydryl reagents, or in the
absence or presence of reducing agents. Amiloride or MIA was used to inhibit sodium transport pathways (sodium/proton exchange) and Ba 21 to inhibit the potassium transport pathways (Virkki and Nikinmaa, 1996; 1998). NPM was used to oxidize any external sulfhydryl groups and ascorbate to reduce cupric ions to cuprous ions. The concentrations of the chemicals were chosen on the basis of earlier data and were 10 mM for MIA, 1 mM for NPM, and 1 mM for ascorbate, (Bogdanova and Nikinmaa, 1998; Parker and Colclasure, 1992). If the above chemicals were used, the cells were preincubated in the media containing the chemical for 15 min before addition of CuSO 4 to the suspension. Na 1, K 1, and Cl 2 fluxes were measured using 22Na, 86Rb, and 36Cl as tracers. Tracer flux measurements in lamprey erythrocytes were carried out as described earlier (Gusev et al., 1992; Gusev and Sherstobitov, 1996; Bogdanova et al., 1998). To study the time course of the effect of CuSO 4 on 36Cl uptake, incubation was started by an addition of cell suspension to the medium containing 100 mM CuSO 4 and the radioactive tracer. One-milliliter aliquots of cell suspension were taken at predetermined time intervals, and 36Cl uptake was terminated by dilution with 10 ml ice-cold washing solution and immediate centrifugation. An aliquot of supernatant was taken to assay radioactivity of the incubation medium, and the cells were washed once more with ice-cold washing saline. Cell pellet was lysed in 0.2 ml 0.6 M HClO 4, and the radioactivity of cell lysate was determined using Wallac 1450 Microbeta Plus liquid scintillation counter. To assay unidirectional Cl 2 influx, the cells were preincubated with CuSO 4 for 2–5 min, 36Cl added, and incubation continued for 3–10 min. Na 1 ( 22Na) uptake and influx measurements were carried out using similar procedure. Radioactivity of cell lysates and incubation medium was measured using a g-counter. Linearity of tracer uptake was always tested in unidirectional flux assay. Unidirectional fluxes were calculated from the equation: J 5 $Ac/Am*@X# e %/t, where Ac and Am are radioactivity of 1 ml of packed cells and 1 ml medium, respectively; [X] e is Cl 2 or Na 1 concentration in the incubation medium, and t is a time of incubation with the tracer. For the measurement of Na 1 efflux, red blood cells were preloaded for 1 h with 22Na (50 mCi/ml) at 20°C and washed free from external tracer, and 22Na efflux was measured for 60 –90 min in the media with and without 100 mM CuSO 4. Aliquots (1 ml) of the cell suspensions were taken every 10 min, and flux was terminated as stated in the influx protocol. The cell pellet was lysed with 1 ml distilled water, and the radioactivity of lysates ( Ac) and total radioactivity of cell suspension ( At) were determined with g-counter. The rate constant of Na 1 efflux was calculated using linear regression analysis of plots of ln[1-( Ac0-Ac9)/At] vs time where Ac9 and Ac0 express the cell radioactivity at the time t9 and t0, respectively. For measurements of unidirectional K 1( 86Rb) influx, the cells were incubated for 5 min in the standard incubation solutions containing 1 mM K 1 and 86 Rb as a label. Net K 1 loss was determined for the cells incubated in K 1-free media for 20 min. K 1 concentration in the medium after incubation as well as intracellular Na 1 and K 1 concentrations were measured by flame photometry (Flapho 40, Karl Zeiss, Jena). To assay intracellular sodium and potassium concentrations, aliquots of cell suspensions (0.1 ml) were washed twice with cold (2– 4°C) washing solution containing (mM) 75 MgCl 2, 85 sucrose, 10 Tris-HCl, and cell pellet lysed in 0.5 ml distilled water. Patch-clamp measurements of conductive cation transport. A small amount of red cell suspension was added to the experimental chamber containing the desired solution, and the cells were allowed to settle for a few minutes before the measurements were started. The bath was grounded through a 3M KCl agar-salt bridge. Patch electrodes were pulled from borosilicate glass capillaries with inner filament (GC150F, Clark Electromedical Instruments) on a two-step Narishige PP-83 puller (Narishige, Japan) and used without heat polishing. Pipette resistance was 6 – 8 MV when filled with pipette internal solution. After seal formation (seal resistance 5–30 GV 2), the membrane patch
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was ruptured by gentle suction. Cell capacitance and series resistance (90%) were compensated for. Whole-cell recordings were made using an EPC-9 amplifier (Heka Electronics, Germany) driven by an Atari Mega STE computer. Voltage protocols designed to obtain current-voltage relations were generated using Atari software. Voltage was defined as the pipette potential with respect to bath (ground). The voltage protocol consisted of alternatingly negative and positive voltage steps in 15-mV increments from the holding potential. Currents were saved directly on hard disk at a sampling rate of 5 kHz after low-pass filtering at 1.5 kHz. The data was analyzed using the program Review (M2 Lab, Instrutech Corporation, Elmont, NY). To study the effect of Cu ions on the conductance of lamprey red cells, CuSO 4 was added to bath (extracellular) solution to a final concentration of 25, 50, or 100 mM. To determine the selectivity of the ion conductance pathways in the presence and absence of copper ions, bath and pipette solutions of different ionic compositions were used. The composition of the bath solution was 127 mM XCl (X denoting K, Na, Li, or NMDG), 1 mM CaCl 2, 10 mM Tris, pH 7.4. The composition of the internal solution was 127 mM NaCl or KCl, 1 mM MgCl 2, 10 mM Tris, pH 7.4. In some experiments, amiloride (1 mM) or MIA (10 mM, final concentration) was added to the bath solutions. Internal solutions were filtered through Millipore filters. Blockers were added and solutions were changed by bath perfusion. Measurements of intracellular and extracellular pH and cell water content. Extracellular pH was monitored using a Beckman pH electrode. To study the effect of copper on external pH, 50 mM CuSO 4 were added to a medium with low buffering capacity (LBM) containing (mM) 150 NaCl, 1 CaCl 2, and 1 HEPES-KOH (pH 7.40 at 20°C). The small amount of buffers helped to stabilize the pH of the solution during the 20-min incubation, but the buffering capacity was small enough that any changes in net fluxes of acid equivalents across the cell membrane could be monitored. In some experiments chloride as a major ion was replaced by either nitrate or bromide using 150 mM NaNO 3 or NaBr instead of NaCl. The pH of the external medium before and after addition of the cells was monitored under constant stirring every 1–2 min. After copper was added, pH of LBM was monitored for 2 min and then the cells were added to maintain final haematocrit about 2–3%. Haemolysis was traced by taking samples of the suspension every 2–5 min. Intracellular pH was measured using the DMO method as described earlier (Nikinmaa and Huestis, 1984). After the red blood cells suspension (Hct 6 – 8%) was preincubated with 14C-DMO (final radioactivity ;740 kBq/ml) for 10 min, the experiment was started by adding 100 mM CuSO 4 (final concentration). Aliquots of the suspension (0.5 ml) were taken into preweighed Eppendorf tubes, and extracellular pH was measured with Radiometer BMS3 Mk2. The incubation medium and cells were then separated by centrifugation (2 min 12,000g), and 0.2 ml supernatant was mixed with 0.2 ml 0.6 M HClO 4 and used to measure the extracellular 14C-DMO activity. Remaining supernatant was carefully removed from the tube containing the cell pellet, the pellet was weighed, and the cells were lysed in 0.2 ml 0.6 M HClO 4. After centrifugation (5 min, 12,000g) a 0.2-ml portion of the supernatant of the cell lysate was used to measure the activity of the 14C-DMO ion in the cell water. Intracellular pH was then calculated from the extracellular pH and the cell/ medium 14C-DMO distribution using the equation
pHi 5 pK DMO 1 log$@DMO# i /@DMO# e *~10ˆ~pHe 2 pKDMO! 1 1! 2 1%,
where pK DMO was taken to be 6.23 at 25°C, and [DMO] i and [DMO] e were the activities of DMO per liter of cell or medium water, respectively. Cell water was determined by weighing the cell pellet, drying it to a constant weight at 80°C and reweighing. Statistical treatments. Results are presented as means and standard errors of the mean. The statistical significance of the differences between the means was calculated using one-way ANOVA followed by LSD test (SPSS software). The values of p , 0.05 were taken as significant.
FIG. 1. Effect of 100 mM CuSO 4 on pH i in lamprey erythrocytes. (A) Time course of CuSO 4 effect. Values are means of 4 independent experiments 6 SEM. (B) The role of Na 1/H 1 exchanger in copper-induced changes in intraerythrocytic pH. The cells were preincubated for 15 min with or without 10 mM MIA before a 10-min incubation with 100 mM CuSO 4. Values are means of 6 independent experiments 6 SEM. Asterisks indicate the statistical significance of the difference between means connected with lines: *p , 0.05, **p , 0.01, ***p , 0.001.
RESULTS
Effect of Copper Ions on Proton Permeability and Intracellular pH of Lamprey Erythrocytes The intracellular pH of lamprey erythrocytes exposed to 100 mM CuSO 4 decreased markedly, from approximately 7.9 before the addition of copper to 7.4 at the end of the 20-min exposure period. (Fig. 1A). The intracellular pH decreased by about 0.024 pH units/min during the copper treatment. The copper-induced intracellular acidification was not abolished by the selective and effective sodium/proton exchange inhibitor MIA (Fig. 1B). To study the effects of copper on the proton permeability of the membrane of lamprey erythrocytes, the cells were equilibrated in weakly buffered medium, and a small bolus of 0.1 M HCl was added. In lamprey erythrocytes the intracellular compartment is effectively isolated from the extracellular compartment owing to the low proton permeability of the membrane, and, therefore, extracellular acid loads cannot be buffered by the effective intracellular buffer, haemoglobin (Nikinmaa and Railo, 1987; Nikinmaa et al., 1995). Thus, after the initial addition of HCl to the extracellular medium, the extracellular pH is reduced and remains low (Fig. 2A). However, when the cells are exposed to CuSO 4 and hydrolysis of the copper salt induces the same level of acidification, the extracellular pH rapidly recovers in halide-containing media (Fig. 2, B and C), indicating that the intracellular buffer is now accessible to the extracellular ions, i.e., that the proton permeability of the cell membrane has markedly increased. Notably, the increase in proton permeability requires the presence of chloride or bromide ions: the pH recovery was not observed in nitrate-containing medium (Fig. 2D).
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reagent N-phenyl maleimide (NPM), which is essentially impermeant and, therefore, only oxidizes extracellularly facing-SH groups, completely inhibited the copper-induced increase in chloride permeability, the flux being 44.4 6 4.2 and 42.8 6 0.73 mmol/L cells/h in copper-treated and control cells, respectively. When NPM-pretreated cells were exposed to 100 mM CuSO 4 in the presence of 1 mM ascorbate, NPM failed to
FIG. 2. Effect of 50 mM CuSO 4 on the permeability of the erythrocyte membrane to proton equivalents. pH o recovery after acidification of weakly buffered media (1 mM HEPES–KOH) of different anion composition was monitored using Beckman pH electrode. Data shown are from representative experiments. (A) Lack of pH o recovery after addition of 0.1 M HCl aliquot into copper-free lamprey erythrocyte suspension, n 5 4. (B–D) pH o recovery after acidification caused by hydrolysis of CuSO 4 added to poorly buffered media. (B) pH o recovery in the standard Cl 2-containing medium, n 5 5. (C) pH o recovery in Br 2-containing medium, n 5 4. (D) pH o recovery in NO 32containing medium, n 5 5. Filled symbols give pH o values in copper-free red cell suspensions, and empty symbols give the values in copper-containing suspensions.
Effect of Copper Ions on Cl 2 Influx Exposure of lamprey erythrocytes to 100 mM copper sulfate caused a pronounced increase in the uptake of Cl 2 in the erythrocytes after an approximately 3-min lag period (Fig. 3A). The maximal rate of Cl 2 influx, 207 6 31 mmol/L cells/h (as compared to 55.8 6 3.2 mmol/L cells/h in control), was measured after a 5-min copper treatment. The result indicates that not only the proton permeability but also the chloride permeability of the red cell membrane is markedly increased by copper. Chloride influx was further increased to 802 6 93 mol/L cells/h already during the first 3–5 min of treatment, when the copper exposure was carried out in the presence of a reducing agent, 1 mmol ascorbate (Fig 3B). Notably, ascorbate alone did not influence the chloride flux, which was 53.7 6 4.3 mmol/L cells/h in ascorbate-treated cells. In contrast, pretreatment of the cells with the sulphydryl
FIG. 3. Effect of CuSO 4 on Cl 2 influx in lamprey erythrocytes. (A) Time course of 36Cl uptake under control conditions (E) in the presence 100 mM CuSO 4 (■) and in the presence of 1 mM ascorbate and 100 mM CuSO 4 (h). Values are means for 5–12 independent experiments 6 SEM. (B) Effect of CuSO 4 on unidirectional Cl 2 influx in the presence of 1 mM ascorbate to 1 mM NPM in the incubation medium. Unidirectional Cl 2 influx was calculated from 10 min 36Cl uptake for control (open bars) and for 2 min tracer uptake in copper-treated cells (hatched bars). Before the tracer was added, the cells were preincubated for 5 min with 100 mM CuSO 4 or for 2 min 1 mM ascorbate 1 100 mM CuSO 4. When 1 mM NPM was used, the cells were pretreated for 15 min before copper were added and then incubated for 10 more min with 100 mM CuSO 4. Asterisks denote the statistical significance of the difference between the means for copper-treated and control cells: ***p , 0.001. Values are means of 5 independent experiments 6 SEM.
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FIG. 5. Effect of CuSO 4 on the erythrocyte water content as a function of time at 5, 10, 15, or 20 min incubation with (■) or without (h) 100 mM CuSO 4. Asterisks denote statistical significance of the difference in mean of the control and copper-treated cells at the same time point, ***p , 0.001. Experiments are means of 6 independent experiments 6 SEM.
copper-sensitive Cl 2 influx already at 10 mM CuSO 4 concentration (at which concentration copper did not affect chloride flux in 155 mM chloride medium; Fig. 4). Effect of Copper Ions on Cell Water and Cation Fluxes FIG. 4. Effect of CuSO 4 on 36Cl uptake in the media of different anion composition. Incubation media contained (mM) NaCl/NaBr/NaNO 3, 140; HEPES–KOH, 5; CaCl 2, 1; glucose, 10. 36Cl uptake was measured during 10 min incubation with the tracer in the absence of copper (open bars), or in the presence of 10 mM CuSO 4 (hatched bars) or 100 mM CuSO 4 (cross-hatched bars). Values are means of 5 independent experiments 6 SEM. ***p , 0.0001 as compared to control.
prevent copper-induced activation of Cl 2 influx (data not shown). All the above data, the simultaneous increase in both proton and chloride transport rates, the effect of reducing agent, and the effect of extracellular sulphydryl modification suggest that cupric ions are reduced to cuprous ions on the extracellular membrane surface, and that these cuprous ions then induce an electroneutral Cl 2/OH 2 exchange across the cell membrane, as has been observed in asolectin and renal brush border membrane vesicles (Karniski, 1992). In asolectin vesicles (Karniski, 1992) replacement of external Cl 2 by Br 2 facilitates Cu 1mediated chloride/hydroxyl exchange. In nitrate-containing medium cuprous ions get spontaneously oxidized back to cupric ions, so no anion exchange should be observed. The effect of anion substitution on the effect of copper on chloride flux was, therefore, tested. As can be seen from Fig. 4, reducing the extracellular chloride concentration to 2 mM and replacing it by nitrate abolished the activation of 36Cl uptake in the presence of 100 mM CuSO 4. In contrast, similar substitution of chloride by bromide resulted in a pronounced activation of the
Exposure of lamprey erythrocytes to 100 mM CuSO 4 results in a significant increase in cellular water content, already within 15 min of exposure (Fig. 5). Copper-induced swelling is associated with dose-dependent increase in cellular Na 1 and TABLE 1 Effect of CuSO 4 on the Lamprey Erythrocyte Na 1 and K 1 Content Intracellular cation concentration, (mmol/L cells) Treatment
Na1
K1
Control 25 mM CuSO 4 50 mM CuSO 4 100 mM CuSO 4 Amiloride, 1 mM 100 mM CuSO 4 1 amiloride Ba 21, 1 mM 100 mM CuSO 4 1 Ba 21
31.3 6 1.6 35.2 6 2.4 37.6 6 1.8* 63.4 6 8.5*** 30.3 6 3.3 33.3 6 4.0 33.5 6 2.4 47.9 6 7.6***
63.1 6 7.1 58.0 6 7.0* 50.4 6 6.1** 44.6 6 6.8*** 63.4 6 6.1 59.6 6 5.2 64.4 6 4.6 46.7 6 8.9***
Note. Cell suspension was treated with 100 mM CuSO 4 for 20 min. Cells were separated by centrifugation and washed free from external Na 1 and K 1 with Mg 21-sucrose solution. The cell pellet was lysed, and cell K 1 and Na 1 content were assayed by flame photometry. Values of 5 independent experiments are means 6 SEM. * p , 0.05. ** p , 0.01. *** p , 0.001 value vs control.
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TABLE 2 Effects of CuSO 4, Ba 21, and Amiloride on Unidirectional K 1 Influx into the Lamprey Erythrocytes K 1 influx, (mmol/L cells/h) Additions
Control
100 mM CuSO 4
None 1 mM Ba 21 1 mM amiloride
1.70 6 0.26 1.06 6 0.11*** 0.99 6 0.12***
1.90 6 0.25 1.67 6 0.26 ### 1.01 6 0.09
Note. The cells were pretreated with the inhibitors for 15 min and then K 1( 86Rb) influx was measured. The presented values 6 SEM are average means of 7 independent experiments. *** A statistically significant difference to the control flux (p , 0.001). ### Significant difference (p , 0.001) as compared to the flux in Ba 21-treated cells in the absence of copper.
decrease in K 1 content (Table 1). Net Na 1 uptake and K 1 loss by the cells exposed to 100 mM CuSO 4 for 20 min is abolished by pretreatment of the cells with 1 mM amiloride, but not with 1 mM Ba 21. To characterize the fluxes involved, we measured both the conductive cation fluxes using the whole cell patch clamp technique and radioactive tracer fluxes in the presence and absence of several ion transport inhibitors. It was previously shown that under physiological conditions the major charge-carrying pathway in lamprey erythrocyte membrane is K 1-selective, with very little current carried by Na 1 or Cl 2 ions (Virkki and Nikinmaa, 1996, 1998). Figure 6 shows a current-voltage relationship as recorded with KCl solution in the pipette and KCl or NaCl media in the bath in the absence or presence of 100 mM CuSO 4. The whole-cell con-
FIG. 6. Effect of 100 mM CuSO 4 on the lamprey erythrocyte membrane conductance to Na 1 and K 1. Whole-cell patch clamp technique was used with KCl solution in the pipette and either NaCl or KCl solutions in the bath. The figure shows representative data of seven experiments.
FIG. 7. Time course of 22Na uptake in lamprey erythrocytes exposed to 100 mM CuSO 4. Copper was added to the cell suspension together with the radioactive tracer. 22Na uptake by copper-treated cells differs significantly ( p , 0.001) from controls already after 3 min of incubation. Values are means 6 SEM of 6 independent experiments.
ductance shows strong inward rectification in symmetric potassium-containing solutions, as observed previously (Virkki and Nikinmaa, 1996). Replacement of K 1 by Na 1 in the bath results in a strong decrease in inward conductance. However, addition of cupric sulphate (25, 50, or 100 mM; n 5 2, 3, and 7, respectively) to the bath solution does not affect either inward or outward whole-cell currents in either sodium- or potassium-containing media. Thus, the possibility of copperinduced activation of any conductive pathway for K 1, Na 1, or Cl 2 can be ruled out. Using sodium-22, we measured the effect of copper on the sodium fluxes. There was a marked increase in both unidirectional influx and efflux of sodium across lamprey erythrocyte membrane after an initial 3–5 min lag period (Fig. 7). In the presence of 100 mM CuSO 4 the rate coefficient for influx was maximally 6.1 6 0.5 h 21, as compared to 0.15 6 0.02 h 21 in control conditions. The rate coefficients for efflux were 11.7 6 1.4 h 21 for copper-exposed cells and 0.89 6 0.18 h 21 in controls. Copper-induced Na 1 efflux was completely abolished by pretreatment with 1 mM amiloride. Dose-response of MIA effect on Na 1 influx in copper-free media gave IC 50 of 2.45 6 0.45 mM with the flux observed after maximal inhibition of 0.075 h 21. However, dramatic increase in Na 1 influx induced after 10 min treatment by 100 mM CuSO 4 was only inhibited by approximately 70% by 1 mM amiloride or 100 mM MIA (Fig. 8). Hence, in addition to stimulation of sodium influx via the sodium/proton exchanger, copper treatment results in activation of the amiloride (MIA)insensitive Na 1 transport pathway. Most likely, the same amiloride-insensitive component of Na 1 influx is preserved when the NPM-pretreated cells are exposed for 10 min to 100 mM CuSO 4. After 15 min pretreatment with 1 mM NPM, Na 1 influx in the presence of copper was 5.01 6 0.11 h 21 whereas
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With regard to potassium transport, exposure to copper resulted in a net potassium loss from the cell. However, the effect of copper ions on the inhibitor sensitivity of potassium fluxes in lamprey erythrocytes depends on the copper concentration used. The potassium efflux induced at 25 mM CuSO 4 was only slightly sensitive to amiloride, but completely inhibited by 1 mM Ba 21 (Fig. 9). In contrast, the potassium efflux induced at 100 mM CuSO 4, was hardly affected by Ba 21 but effectively inhibited by amiloride. Although copper exposure affected the net loss of potassium from the cells markedly, it did not influence the influx of potassium (as tested by measuring the unidirectional 86Rb influx) at 100 mM CuSO 4 (Table 2) but prevented Ba 21-induced inhibition of K 1 influx. DISCUSSION
FIG. 8. Effect of the inhibitors of Na 1/H 1 exchanger on copper-activated Na 1 influx. The cells were preincubated for 15 min with either MIA (10 or 100 mM) or amiloride (1 mM) before the addition of 100 mM CuSO 4. Radioactive tracer was added 5 min after the toxicant and uptake followed for 2 min. Flux values in copper-treated cells differ significantly ( p , 0.001) from controls regardless of inhibitors’ application. Values are means of 5 experiments 6 SEM.
in copper-free NPM-pretreated cells it was 0.156 6 0.016 h 21. After replacement of external Cl 2 by NO 32, 10 min treatment with 100 mM CuSO 4 resulted in increase in Na 1 influx from 0.198 6 0.028 h 21 to 1.79 6 0.12 h 21 compared to 0.156 6 0.008 h 21 vs 9.09 6 0.23 h 21 in the chloride-containing medium without and with CuSO 4, respectively. In bromide-containing saline 100 mM CuSO 4 increased Na 1 influx from 0.116 6 0.0037 to 13.5 6 0.36 h 21. Thus, replacement of extracellular Cl 2 by nitrate, preventing copper-induced increase in proton permeability, reduced the sodium influx to the same level as inhibition of the sodium/proton exchanger by MIA. It indicates that the copper-induced increase in sodium/ proton exchange activity is most likely secondary to the changes in intracellular pH. Moreover, an unidentified Na 1 transport pathway is induced by cupric ions in parallel to Na 1/H 1 exchange activation, since NPM pretreatment is not abolishing its activation by CuSO 4.
We used high bulk concentrations of copper sulfate to induce membrane level disturbances in lamprey erythrocytes rapidly. Actual concentration of free cupric ions in the medium was not measured but did not exceed 10 210 M due to high affinity of Cu 21 ions to such ligands as chloride, hydroxyl, and Tris ions, as follows from complex stability constants given by Smith and Martell (1976). The observed disturbances, cell swelling, net ion losses, and pH disturbances are similar to those observed in Ehrlich ascites tumor cells treated with CuSO 4 (Kramhoft et al., 1988; Lambert et al., 1984). Thus, at the membrane level, it is possible that the mechanisms implicated for lamprey erythrocytes are important behind the toxic effects in other cell types and also in vivo. Two observations suggest that, initially, the divalent form of
FIG. 9. Net K 1 loss into K 1-free incubation medium from the lamprey erythrocytes exposed to either 25 or 100 mM CuSO 4 for 20 min. 1 mM BaCl 2 (hatched bars) or 1 mM amiloride (cross-hatched bars) was added to the cell suspension 15 min prior to copper sulphate. Open bars represent K 1 loss from the copper-treated cells in the absence of inhibitors. Asterisks denote statistical difference (*p , 0.05 and ***p , 0.001) of K 1 loss as compared to potassium loss from the cells into copper-free medium (dashed line). Values are means of 6 independent experiments 6 SEM.
COPPER EFFECTS ON ION TRANSPORT
copper is reduced to a univalent form which causes the membrane level effects. First, the initial lag period in the copper effect on ion fluxes disappears in the presence of the reducing agent, ascorbate, and, second, ascorbate accentuates the effects of copper on the measured parameters. Extracellularly facing sulphydryl groups appear to be involved in the reduction of divalent copper to monovalent copper, because their oxidation to –S–S– bonds fully inhibits the copper-induced activation of Cl 2 influx. Notably, reduction of cupric ions to cuprous ions is an important step of copper uptake in a number of cell types including mammalian hepatocytes, C-6 and K562 cell lines, and in yeast cells (Qian et al., 1995; Davidson et al., 1994; Hasset and Kosman, 1995). In the plasma membrane of rat hepatocytes the electron transfer required for the formation of cupric ions is catalyzed by NADH oxidase (Van den Berg and McArdle, 1994), an enzyme which is likely to be present also on lamprey red cell membrane. In the yeast Saccharomyces cerevisiae, cell surface cupric reductases Fre1p and Fre2p are involved in the facilitation of copper transport across the membrane (Georgatsou et al., 1997). Cuprous ions form relatively poorly water-soluble, and thus lipophilic, ion pairs with halides (chloride and bromide) and hydroxyl ion. These ion pairs incorporate into the lipid bilayer and mediate electroneutral halide/hydroxyl ion exchange, thus increasing the apparent halide and proton (or actually hydroxyl ion) permeability. This conclusion is supported by the observation that the chloride and proton (hydroxyl ion) permeabilities are reduced with increasing water solubility (decreasing lipid solubility) of the ion pair: the effect is greater with bromide with a solubility constant of 4.15p10 28 for CuBr than with chloride with a solubility constant of 1.02p10 26 mol/L for CuCl. Since Cu 1 is unstable in nitrate-containing medium, ion pair formation does not occur and, thus, no changes in anion (or apparent proton) permeability take place. Univalent copper ions also induce electroneutral chloride/hydroxyl ion exchange across model membranes and across renal brush border membrane vesicles (Karniski, 1992). Halide/hydroxyl ion exchange across plasma membranes and lipid bilayers via similar mechanism is also induced by tributyltin, Hg 21, and Tl 31 (Gutknecht, 1981; Wieth and Tosteson, 1979; Karniski, 1992; Dias and Monreal, 1994). In addition to the mechanism presented above, the copperinduced increase in proton permeability could be caused by primary activation of the sodium/proton exchange, the major proton transporting pathway of lamprey erythrocytes (Virkki and Nikinmaa, 1994), as has been described for Ehrlich ascites tumor cells (Kramhoft et al., 1988). However, this is not the case for lamprey erythrocytes. With the proton and sodium gradients present across lamprey erythrocyte membrane (Virkki and Nikinmaa, 1994), activation of the sodium/proton exchange by copper would have resulted in intracellular alkalinization instead of the pronounced acidification observed. Furthermore, the simultaneous increase in both the intracellular
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proton and the sodium activity is incompatible with direct effects of copper on the sodium/proton exchanger. On the other hand, the copper-induced increase in the sodium permeability appears to be largely due to a secondary activation of the sodium/proton exchange as a consequence of the initial cellular acidification which is a powerful stimulus for the activation of the sodium/proton exchange in lamprey erythrocytes (Virkki and Nikinmaa, 1994). The increase in sodium influx is markedly inhibited by MIA, and the amiloride-sensitive sodium flux is much less increased by copper treatment in nitrate-containing medium (in which acidification is not observed) than in chloride containing medium. A minor proportion of the copper-induced increase in sodium permeability is due to an amiloride-insensitive sodium transport pathway which does not require intracellular acidification for activation and is sensitive to extracellular divalent copper ions. In molluscan neurons 1–100 mM Cu 21, 100 mM Hg 21, or Ag 1 induced rapid irreversible depolarization accompanied by an increase in membrane conductance for Na 1 (Weinreich and Wonderlich, 1987). However, based on our patch-clamp data, it is unlikely that the transport pathway activated in lamprey would be a channel, since the ion conductivity is not affected by copper treatment. With regard to potassium transport, copper did not affect membrane premeability. However, there was a net potassium loss from copper-exposed erythrocytes which at low copper concentrations (25–50 mM) probably occurred via the Ba 21sensitive, low conductance K 1 channel (Virkki and Nikinmaa, 1996), and at the higher copper concentration (100 mM) via the Ba 21-insensitive, swelling-activated channel (Virkki and Nikinmaa, 1998). Inhibitory action of Cu 21 on Na 1/K 1 pump has been reported for many cell types (e.g., Li et al., 1996). However, since copper ions did not affect K 1 influx, Na 1/K 1 ATPase was not inhibited in lamprey erythrocytes in the present experiments. Notably, the effects described in the present study are mainly caused by extracellular copper ions. The inhibition of Na 1/K 1 ATPase, on the other hand, requires that Cu 21 ions prevent interaction of the enzyme with Mg 21 in the cytosolic side of the membrane (Li et al., 1996). We have not investigated rates of copper transport into the lamprey erythrocyte. However, low transport rates for copper ions can be expected, since in erythrocytes copper transport normally occurs mainly via the anion exchange pathway (Alda and Garay, 1988), and lamprey erythrocytes are characterized by a virtual absence of anion exchange (e.g., Nikinmaa and Railo, 1987; Tufts and Boutilier, 1989). In conclusion, the membrane effects of copper ions appear to involve the following sequence of events. (1) Cu 21 is reduced to Cu 1 with integral protein having essential externally facing SH-groups catalyzing the reaction. (2) Consequently, poorly water-soluble electroneutral ion pairs CuCl or CuOH incorporate into the lipid bilayer and shuttle between the internal and external surface of the membrane inducing a rapid Cl 2/OH 2
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exchange. (3) Protonophore-like action of Cu 1 results in dissipation of the transmembrane proton gradient and marked intracellular acidification. (4) Decrease in pH i activates the amiloride-sensitive Na 1/H 1 exchange. (5) The net uptake of chloride and sodium cause the cell swelling, and, consequently, net potassium efflux occurs mainly via the swelling-activated pathways. In addition, interaction of cupric ions with the membrane activates an amiloride-insensitive Na 1 transport pathway of unknown nature. The data of the present study and the proposed mechanisms for copper-induced effects are compatible with earlier results on the toxic effects of copper, i.e., osmolarity disorders (Heath, 1997), net loss of ions and acidosis observed in gills (Wilson and Taylor, 1993), collapse of transmembrane proton gradient on mitochondrial membrane after penetration of copper ions into the cells (Wojczak et al., 1996), and net K 1 loss from copper-treated yeast cells (Passow and Rothstein, 1960). The interesting parallel between the effects of copper and the effects of organic tin chlorides suggest that the wide use of copper salts in agriculture as fungicide, as a food preservative, and for textile dyeing and metal coloring should be carefully monitored with regard to the toxic effects. ACKNOWLEDGMENTS
Gusev, G. P., and Sherstobitov, A. O. (1996). An amiloride-sensitive, volumedependent Na 1 transport across the lamprey (Lampetra fluviatilis) erythrocyte membrane. Gen. Physiol. Biophys. 15, 129 –143. Gutknecht, J. (1981). Inorganic mercury (Hg 21) transport through lipid bilayer membrane, J. Membr. Biol. 61, 61– 66. Gwozdzinski, K. (1991). A spin label study of the action of cupric and mercuric ions on human red blood cells. Toxicology 65, 315–323. Hansen, H. J. M., Olsen, A. G., and Rosenkilde, P. (1996). The effect of Cu on gill and esophagus lipid metabolism in the rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 113C, 23–29. Harris, E., and Giltin, J. D. (1996). Genetic and molecular basis for copper toxicity. Am. J. Clin. Nutr. 63, 836S– 841S. Hassett, R., and Kosman, D. J. (1995). Evidence for Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae. J. Biol. Chem. 270, 128 –134. Heath, A. G. (1997). Water Pollution and Fish Physiology. CRC Press, Boca Raton, FL. Karniski, L. P. (1992). Hg 21 and Cu 1 are ionophores, mediating Cl 2/OH 2 exchange in liposomes and rabbit renal brush border membranes. J. Biol. Chem. 267, 19218 –19225. Kramhoft, B., Lambert, I. H., and Hoffmann, E. K. (1988). Na 1/H 1 exchange in Ehrlich ascites tumour cells: Activation by cytoplasmic acidification and by treatment with cupric sulphate. J. Membr. Biol. 102, 35– 48. Lambert, I. H., Kramhoft, B., and Hoffmann, E. K. (1984). Effect of copper on volume regulation in Ehlich ascites tumour cells. Mol. Physiol. 6, 83–98. Li, J., Lock, R. A. C., Klaren, P. H. M., Swarts, H. G. P., Schuurmans Stekhoven, F. M. A. H., Wendelaar Bonga, S. E., and Flik, G. (1996). Kinetics of Cu 21 inhibition of Na 1/K 1 ATPase, Toxicol. Lett. 87, 31–38.
This work was supported by Academy of Finland, Research Council for the Environment and Natural Resources (grant 40830) for M.N., and CIMO scholarship and Hali-Gali Club scholarship for A. B. Riitta Niemi is acknowledged for excellent technical assistance and A. A. Lew and A. O. Sherstobitov for valuable discussions.
Nikinmaa, M., and Huestis, W. H. (1984). Adrenergic swelling in nucleated erythrocytes: cellular mechanisms in a bird, domestic goose and two teleosts, striped bass and rainbow trout. J. Exp. Biol. 113, 215–224.
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Nikinmaa, M., and Railo, E. (1987). Anion movements across lamprey (Lamptera fluviatilis) red cell membrane. Biochim. Biophys. Acta 899, 134 –136.
Alda, J. O., and Garay, R. (1990). Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells. Am. J. Physiol. 259, C570 –C576. Bogdanova, A. Y., Sherstobitov, A. O., and Gusev, G. P. (1998). Chloride transport in red blood cells of lamprey Lampetra fluviatilis: Evidence for a novel anion-exchange system. J. Exp. Biol. 201, 693–700. Bogdanova, A. Y., and Nikinmaa, M. (1998). Dehydroabietic acid (DHAA), a major effluent component of paper and pulp industry, decreases erythrocyte pH in lamprey (Lampetra fluviatilis). Aquat. Toxicol. 43, 111–120.
Nikinmaa, M., Kunnamo-Ojala, T., and Railo, E. (1986). Mechanisms of pH regulation in lampery (Lampetra fluviatilis) red blood cells. J. Exp. Biol. 122, 355–367.
Nikinmaa, M., Airaksinen, S., and Virkki, L. V. (1995). Haemoglobin function in intact lamprey erythrocytes: interactions with membrane with membrane function in the regulation of gas transport and acid-base balance. J. Exp. Biol. 198, 2423–2430. Onishi, S. T., and Asai, H. (1985). Lamprey erythrocytes lack glycoproteins and anion transport. Comp. Biochem. Physiol. 81B, 405– 407. Passow, H., and Rothstein, A. (1960). The binding of mercury by the yeast cell in relation to changes in permeability. J. Gen. Physiol. 43, 621– 633.
Davidson, L. A., McOrmond, S. L., and Harris, E. D. (1994). Characterization of a particulate pathway for copper in K562 cells. Biochim. Biophys. Acta 1221, 1– 6.
Parker, J. C., and Colclasure, G. C. (1992). Action of thiocyanate and Nphenilmaleimide on volume-responsive Na 1 and K 1 transport in dog red cells. Am. J. Physiol. 262, C418 –C421.
Dias, R. S., and Monreal, J. (1994). Thallium mediates a rapid chloride/ hydroxyl exchange through myelin lipid bilayers. Mol. Pharmacol. 46, 1210 –1216.
Piriou, A., Tallineau, C., Chahboun, S., Pontcharraud, R., and Guillard, O. (1987). Copper induced lipid peroxidation and hemolysis in whole blood: evidence for a lack of correlation, Toxicology 47, 351–361.
Fairbanks, V. F. (1967). Copper sulfate-induced hemolytic anemia. Arch. Intern. Med. 120, 428 – 432.
Rabergh, C. M. I., Isomaa, B., and Eriksson, J. E. (1992). The resin acids dehydroabietic acid and isopimaric acid inhibit bile acid uptake and perturb potassium transport in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss), Aquat Toxicol. 23, 169 –180.
Georgatsou, E., Mavrogiannis, L. A., Fragiadakis, G. S., and Alexandraki, D. (1997). The yeast Fre1p-Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator. J. Biol. Chem. 272, 13786 –13792. Gusev, G. P., Sherstobitov, A. O., and Bogdanova, A. Yu. (1992). Sodium transport in red cells of lamprey Lampetra fluviatitls. Comp. Biochem. Physiol. 101A, 569 –572.
Qian, Y.-C., Tiffany-Castiglioni, E., and Harris, E. (1995). Copper transport and kinetics in cultured rat C6 glioma cells. Am. J. Physiol. 269, C892– C898. Selwyn, M. J., Dawson, A. P., Stockdale, M., and Gains, N. (1970). Chloridehydroxide exchange across mitochondrial, erythrocyte and artificial mem-
COPPER EFFECTS ON ION TRANSPORT branes mediated by trialkyl- and triphenyltin compounds. Eur. J. Biochem. 14, 120 –126. Smith, R. M., and Martell, A. E., Ed. (1976). Critical Stability Constants. Vol. 4, Plenum Press, New York. Tufts, B. L., and Boutilier, R. G. (1989). The absence of rapid chloride/ bicarbonate exchange in lamprey erythrocytes: Implications for CO 2 transport and ion distribution between plasma and erythrocytes in the blood of Petromyson marinus. J. Exp. Biol. 144, 565–576. Van den Berg, G. J., and McArdle, H. J. (1994). A plasma membrane NADH oxidase is involved in copper uptake by plasma membrane vesicles isolated form rat liver. Biochim. Biophys. Acta 1195, 276 –280. Virkki, L., and Nikinmaa, M. (1994). Activation and physiological role of Na 1/H 1 exchange in lamprey (Lampetra fluviatilis) erythrocytes. J. Exp. Biol. 191, 89 –105. Virkki, L., and Nikinmaa, M. (1996). Conductive ion transport across the erythrocyte membrane of lamprey (Lampeta fluviatilis) erythrocytes in iso-
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tonic conditions is mainly via an inwardly rectifying K 1 channel. Comp. Biochem. Physiol. 115A, 169 –176. Virkki, L., and Nikinmaa, M. (1998). Two distinct K 1 channels in lamprey (Lampetra fluviatilis) erythrocyte membrane characterized by single channel patch clamp. J. Membrane Biol. 163, 47–53. Weinreich, D., Wonderlin, W. F. (1987). Copper activates a unique inward current in molluscan neurones. J. Physiol. 394, 429 – 443. Wieth, J. O., and Tosteson, M. T. (1979). Organotin-mediated exchange diffusion of anions in human red cells. J. Gen. Physiol. 73, 765–788. Wilson, R. W., and Taylor, E. W. (1993). The physiological responses of freshwater rainbow trout, Oncorhynchus mykiss, during acutely lethal copper exposure. J. Comp. Physiol. 163B, 38 – 47. Wojczak, L., Nikitina, E. R., Czyz, A., and Skulskii, I. A. (1996). Cuprous ions activate glibenclamide-sensitive potassium channel in liver mitochondria. Biochem. Biophys. Res. Commun. 223, 468 – 473.
Copper Effects on Ion Transport across Lamprey Erythrocyte Membrane: Cl 2/OH 2 Exchange Induced by Cuprous Ions Anna Y. Bogdanova,* Leila V. Virkki,† Gennadii P. Gusev,‡ and Mikko Nikinmaa* *Department of Biology, University of Turku, FIN-20014 Turku, Finland; †Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520; and ‡Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Received January 28, 1999; accepted June 17, 1999
Copper Effects on Ion Transport across Lamprey Erythrocyte Membrane: Cl 2/OH 2 Exchange Induced by Cuprous Ions. Bogdanova, A. Y., Virkki, L. V., Gusev, G. P., and Nikinmaa, M. (1999). Toxicol. Appl. Pharmacol. 159, 204 –213. We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO 4 concentration of 100 mM caused anion-dependent intracellular acidification and increase in Cl 2 influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lagphase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na 1 fluxes in both directions and a net Na 1 uptake. Copper-induced Na 1 transport was partially amiloride(MIA)-sensitive representing Na 1/H 1 exchange. The nature of the amiloride-insensitive fraction of copper-activated Na 1 influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K 1 and Cl 2 volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na 1/H 1 exchange and consecutive volume changes cause the RVD response. © 1999 Academic Press
Key Words: copper; intracellular pH; chloride-hydroxyl exchange.
Although copper is an essential trace element for all living organisms, its ionic forms are highly toxic (Harris and Giltin, 1996; Heath, 1997). Since Cu(II) is the dominant form of the element in oxygen-equilibrated aqueous solutions, most of the studies on the copper toxicology have concentrated on the effects of the cupric ions both on the organismic and cellular level. However, within the organism the partial pressure of oxygen is much lower than in the ambient atmosphere, and copper exists mostly as metal–protein complexes. The latter 0041-008X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
conditions may favor formation and stabilization of cuprous ions. Thus, it remains uncertain as to which copper species exerts the toxic effects in the organism. Being the first contact site between the metal ions and the cell, cell membranes may be the main target of copper action. In fishes exposed to sublethal copper concentrations net loss of ions from the organism results from a disturbance in the function of gill epithelial membranes (Wilson and Taylor, 1993; Hansen et al., 1996). In humans, copper-induced haemolytic anemia results from the destruction of erythrocyte membranes (Fairbanks, 1967; Piriou et al., 1987). Both cupric and cuprous ions are reported to affect membrane function. Divalent copper inhibits Na 1/K 1 ATPase in several different cell types including erythrocytes (Li et al., 1996) hence causing a slow dissipation of transmembrane K 1 and Na 1 gradients. Copper (II) ions also induce an increase in sodium conductivity in human erythrocytes (Gwozdzinski, 1991) and molluscan neurons (Weinreich and Wonderlich, 1987). This speeds up the copper-induced depolarization. Furthermore, cupric ions stimulate Na 1/H 1 exchanger in Ehrlich ascites tumor cells (Kramhoft et al., 1988). In asolectin as well as in renal brush border vesicles treated with micromolar concentrations of cuprous sulphate (stabilized by addition of ascorbate as reducing agent), Karniski (1992) observed a rapid copper-mediated electroneutral Cl 2/OH 2 exchange driven by transmembrane pH gradient. He suggested that cuprous ions act similarly to organometal compounds such as tributyltin incorporating into the lipid bilayer in the form of uncharged pairs with halides (Selwin et al., 1970; Wieth and Tosteson, 1979). At present, it is not known how all the above mechanisms of copper action on cellular ion transport are interrelated. The effects of copper ions on membrane transport can be conveniently studied using lamprey erythrocytes. These cells lack rapid band 3-mediated anion exchange (Onishi and Asai, 1985; Nikinmaa and Railo, 1987; Tufts and Boutilier, 1989; Bogdanova et al., 1998), whereby the copper-induced changes in chloride permeability can easily be monitored. The lack of the anion exchange, furthermore, makes it possible to study primarily the effects of extracellular copper, since Cu 21 uptake in, e.g., human erythrocytes, proceeds mainly via the anion
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exchanger (Alda and Garay, 1990). Lamprey erythrocytes have functional Na 1/K 1-ATPase and amiloride-sensitive Na 1/H 1 exchanger (Nikinmaa et al., 1986; Gusev et al., 1992; Virkki and Nikinmaa, 1994) thus enabling the study of all the reported major targets of copper action in an integrative fashion. We have used lamprey erythrocytes to study (1) the effects of copper ions on the transport of chloride, acid equivalents, sodium and potassium, and consecutive changes in the cell volume using radioactive tracers and patch-clamp technique, (2) the interrelationships between the changes in ion fluxes using ion substitutions and specific inhibitors, and (3) the role played by univalent and divalent copper ions on the membrane level studying copper effects in the presence of sulfhydryl reagents, reducing agents, and varying anion composition of the incubation medium. MATERIALS AND METHODS Chemicals. Stock solutions of CuSO 4 and BaCl 2 were prepared in distilled water. Amiloride (Sigma, St. Louis, USA), 5-(N-methyl N-isobuthyl) amiloride, (MIA, Research Biochemicals International, Natick, USA), and R(1)dihydroindenyloxyalkanoic acid (R(1)-DIOA, Research Biochemicals International, Natick, USA) were dissolved in dimethyl sulfoxide (DMSO), and appropriate amounts of solvent were always added to the controls. N-phenyl maleimide (NPM) is from Sigma. 5,5-Dimethyl-[2- 14C]-oxazolidene-2,4-dione (DMO), 22Na, 36Cl, and 86Rb were obtained from Amersham International (Buckinghamshire, England) or Isotope (St. Petersburg, Russia) and dissolved in distilled water at known specific activities. Ascorbate-containing solutions were prepared fresh before use. Animal handling and experimental procedures. River lampreys (Lampetra fluviatilis, 25–100 g) were maintained in laboratory conditions at 5°C for a minimum of 1 week before their red cells were used for experiments. The lampreys were anaesthetized with n-ethyl amino benzoic acid ethyl ester (1 g/L), and blood samples were drawn from the caudal vessel into heparinized syringes. Cells were then pelleted, washed with a solution containing 154 mM NaCl and 1 mM KCl, and finally resuspended in standard incubation medium containing (in mM) 144 NaCl, 1 KCl, 10 Tris–HCl, 10 glucose (pH 7.4 at 20°C). All the experiments were carried out at 20°C. Final haematocrit of the cell suspension was 3–5% if not stated otherwise. In a preliminary set of experiments the highest sublytic copper concentration was determined by measuring the extracellular haemoglobin concentration after separating the incubation medium and cells by centrifugation. Treatment of lamprey erythrocytes with 100 mM CuSO 4 caused hemolysis which started after 20 –30 min of exposure. At higher copper concentrations extensive hemolysis was observed in less than 20 min of treatment. Thus, the highest concentration of copper used in the experiments was 100 mM. Since even at this concentration, hemolysis was observed during prolonged incubations, it was critical for the interpretation of our results that the integrity of the cell membrane was not disrupted during the time course of the experiments described. For this reason, we excluded all the incubations in which significant hemolysis occurred. The lack of hemolysis indicates that no macromolecular leakage from the cells occurs. Further, we measured unidirectional rubidium fluxes (as a tracer for potassium) across the cell membrane. An increase in potassium permeability is often taken to indicate disturbances in membrane integrity (e.g., Rabergh et al., 1992). In the present experiments, 100 mM copper concentration did not affect the rubidium permeability (Table 2). Together, the lack of copper effects on rubidium permeability and on macromolecular leakage indicate that the membrane integrity was not compromised. Measurements of Na 1, K 1, and Cl 2 uptake and unidirectional fluxes. The measurements were carried out either in the absence or presence of transport inhibitors, in the absence or presence of sulfhydryl reagents, or in the
absence or presence of reducing agents. Amiloride or MIA was used to inhibit sodium transport pathways (sodium/proton exchange) and Ba 21 to inhibit the potassium transport pathways (Virkki and Nikinmaa, 1996; 1998). NPM was used to oxidize any external sulfhydryl groups and ascorbate to reduce cupric ions to cuprous ions. The concentrations of the chemicals were chosen on the basis of earlier data and were 10 mM for MIA, 1 mM for NPM, and 1 mM for ascorbate, (Bogdanova and Nikinmaa, 1998; Parker and Colclasure, 1992). If the above chemicals were used, the cells were preincubated in the media containing the chemical for 15 min before addition of CuSO 4 to the suspension. Na 1, K 1, and Cl 2 fluxes were measured using 22Na, 86Rb, and 36Cl as tracers. Tracer flux measurements in lamprey erythrocytes were carried out as described earlier (Gusev et al., 1992; Gusev and Sherstobitov, 1996; Bogdanova et al., 1998). To study the time course of the effect of CuSO 4 on 36Cl uptake, incubation was started by an addition of cell suspension to the medium containing 100 mM CuSO 4 and the radioactive tracer. One-milliliter aliquots of cell suspension were taken at predetermined time intervals, and 36Cl uptake was terminated by dilution with 10 ml ice-cold washing solution and immediate centrifugation. An aliquot of supernatant was taken to assay radioactivity of the incubation medium, and the cells were washed once more with ice-cold washing saline. Cell pellet was lysed in 0.2 ml 0.6 M HClO 4, and the radioactivity of cell lysate was determined using Wallac 1450 Microbeta Plus liquid scintillation counter. To assay unidirectional Cl 2 influx, the cells were preincubated with CuSO 4 for 2–5 min, 36Cl added, and incubation continued for 3–10 min. Na 1 ( 22Na) uptake and influx measurements were carried out using similar procedure. Radioactivity of cell lysates and incubation medium was measured using a g-counter. Linearity of tracer uptake was always tested in unidirectional flux assay. Unidirectional fluxes were calculated from the equation: J 5 $Ac/Am*@X# e %/t, where Ac and Am are radioactivity of 1 ml of packed cells and 1 ml medium, respectively; [X] e is Cl 2 or Na 1 concentration in the incubation medium, and t is a time of incubation with the tracer. For the measurement of Na 1 efflux, red blood cells were preloaded for 1 h with 22Na (50 mCi/ml) at 20°C and washed free from external tracer, and 22Na efflux was measured for 60 –90 min in the media with and without 100 mM CuSO 4. Aliquots (1 ml) of the cell suspensions were taken every 10 min, and flux was terminated as stated in the influx protocol. The cell pellet was lysed with 1 ml distilled water, and the radioactivity of lysates ( Ac) and total radioactivity of cell suspension ( At) were determined with g-counter. The rate constant of Na 1 efflux was calculated using linear regression analysis of plots of ln[1-( Ac0-Ac9)/At] vs time where Ac9 and Ac0 express the cell radioactivity at the time t9 and t0, respectively. For measurements of unidirectional K 1( 86Rb) influx, the cells were incubated for 5 min in the standard incubation solutions containing 1 mM K 1 and 86 Rb as a label. Net K 1 loss was determined for the cells incubated in K 1-free media for 20 min. K 1 concentration in the medium after incubation as well as intracellular Na 1 and K 1 concentrations were measured by flame photometry (Flapho 40, Karl Zeiss, Jena). To assay intracellular sodium and potassium concentrations, aliquots of cell suspensions (0.1 ml) were washed twice with cold (2– 4°C) washing solution containing (mM) 75 MgCl 2, 85 sucrose, 10 Tris-HCl, and cell pellet lysed in 0.5 ml distilled water. Patch-clamp measurements of conductive cation transport. A small amount of red cell suspension was added to the experimental chamber containing the desired solution, and the cells were allowed to settle for a few minutes before the measurements were started. The bath was grounded through a 3M KCl agar-salt bridge. Patch electrodes were pulled from borosilicate glass capillaries with inner filament (GC150F, Clark Electromedical Instruments) on a two-step Narishige PP-83 puller (Narishige, Japan) and used without heat polishing. Pipette resistance was 6 – 8 MV when filled with pipette internal solution. After seal formation (seal resistance 5–30 GV 2), the membrane patch
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was ruptured by gentle suction. Cell capacitance and series resistance (90%) were compensated for. Whole-cell recordings were made using an EPC-9 amplifier (Heka Electronics, Germany) driven by an Atari Mega STE computer. Voltage protocols designed to obtain current-voltage relations were generated using Atari software. Voltage was defined as the pipette potential with respect to bath (ground). The voltage protocol consisted of alternatingly negative and positive voltage steps in 15-mV increments from the holding potential. Currents were saved directly on hard disk at a sampling rate of 5 kHz after low-pass filtering at 1.5 kHz. The data was analyzed using the program Review (M2 Lab, Instrutech Corporation, Elmont, NY). To study the effect of Cu ions on the conductance of lamprey red cells, CuSO 4 was added to bath (extracellular) solution to a final concentration of 25, 50, or 100 mM. To determine the selectivity of the ion conductance pathways in the presence and absence of copper ions, bath and pipette solutions of different ionic compositions were used. The composition of the bath solution was 127 mM XCl (X denoting K, Na, Li, or NMDG), 1 mM CaCl 2, 10 mM Tris, pH 7.4. The composition of the internal solution was 127 mM NaCl or KCl, 1 mM MgCl 2, 10 mM Tris, pH 7.4. In some experiments, amiloride (1 mM) or MIA (10 mM, final concentration) was added to the bath solutions. Internal solutions were filtered through Millipore filters. Blockers were added and solutions were changed by bath perfusion. Measurements of intracellular and extracellular pH and cell water content. Extracellular pH was monitored using a Beckman pH electrode. To study the effect of copper on external pH, 50 mM CuSO 4 were added to a medium with low buffering capacity (LBM) containing (mM) 150 NaCl, 1 CaCl 2, and 1 HEPES-KOH (pH 7.40 at 20°C). The small amount of buffers helped to stabilize the pH of the solution during the 20-min incubation, but the buffering capacity was small enough that any changes in net fluxes of acid equivalents across the cell membrane could be monitored. In some experiments chloride as a major ion was replaced by either nitrate or bromide using 150 mM NaNO 3 or NaBr instead of NaCl. The pH of the external medium before and after addition of the cells was monitored under constant stirring every 1–2 min. After copper was added, pH of LBM was monitored for 2 min and then the cells were added to maintain final haematocrit about 2–3%. Haemolysis was traced by taking samples of the suspension every 2–5 min. Intracellular pH was measured using the DMO method as described earlier (Nikinmaa and Huestis, 1984). After the red blood cells suspension (Hct 6 – 8%) was preincubated with 14C-DMO (final radioactivity ;740 kBq/ml) for 10 min, the experiment was started by adding 100 mM CuSO 4 (final concentration). Aliquots of the suspension (0.5 ml) were taken into preweighed Eppendorf tubes, and extracellular pH was measured with Radiometer BMS3 Mk2. The incubation medium and cells were then separated by centrifugation (2 min 12,000g), and 0.2 ml supernatant was mixed with 0.2 ml 0.6 M HClO 4 and used to measure the extracellular 14C-DMO activity. Remaining supernatant was carefully removed from the tube containing the cell pellet, the pellet was weighed, and the cells were lysed in 0.2 ml 0.6 M HClO 4. After centrifugation (5 min, 12,000g) a 0.2-ml portion of the supernatant of the cell lysate was used to measure the activity of the 14C-DMO ion in the cell water. Intracellular pH was then calculated from the extracellular pH and the cell/ medium 14C-DMO distribution using the equation
pHi 5 pK DMO 1 log$@DMO# i /@DMO# e *~10ˆ~pHe 2 pKDMO! 1 1! 2 1%,
where pK DMO was taken to be 6.23 at 25°C, and [DMO] i and [DMO] e were the activities of DMO per liter of cell or medium water, respectively. Cell water was determined by weighing the cell pellet, drying it to a constant weight at 80°C and reweighing. Statistical treatments. Results are presented as means and standard errors of the mean. The statistical significance of the differences between the means was calculated using one-way ANOVA followed by LSD test (SPSS software). The values of p , 0.05 were taken as significant.
FIG. 1. Effect of 100 mM CuSO 4 on pH i in lamprey erythrocytes. (A) Time course of CuSO 4 effect. Values are means of 4 independent experiments 6 SEM. (B) The role of Na 1/H 1 exchanger in copper-induced changes in intraerythrocytic pH. The cells were preincubated for 15 min with or without 10 mM MIA before a 10-min incubation with 100 mM CuSO 4. Values are means of 6 independent experiments 6 SEM. Asterisks indicate the statistical significance of the difference between means connected with lines: *p , 0.05, **p , 0.01, ***p , 0.001.
RESULTS
Effect of Copper Ions on Proton Permeability and Intracellular pH of Lamprey Erythrocytes The intracellular pH of lamprey erythrocytes exposed to 100 mM CuSO 4 decreased markedly, from approximately 7.9 before the addition of copper to 7.4 at the end of the 20-min exposure period. (Fig. 1A). The intracellular pH decreased by about 0.024 pH units/min during the copper treatment. The copper-induced intracellular acidification was not abolished by the selective and effective sodium/proton exchange inhibitor MIA (Fig. 1B). To study the effects of copper on the proton permeability of the membrane of lamprey erythrocytes, the cells were equilibrated in weakly buffered medium, and a small bolus of 0.1 M HCl was added. In lamprey erythrocytes the intracellular compartment is effectively isolated from the extracellular compartment owing to the low proton permeability of the membrane, and, therefore, extracellular acid loads cannot be buffered by the effective intracellular buffer, haemoglobin (Nikinmaa and Railo, 1987; Nikinmaa et al., 1995). Thus, after the initial addition of HCl to the extracellular medium, the extracellular pH is reduced and remains low (Fig. 2A). However, when the cells are exposed to CuSO 4 and hydrolysis of the copper salt induces the same level of acidification, the extracellular pH rapidly recovers in halide-containing media (Fig. 2, B and C), indicating that the intracellular buffer is now accessible to the extracellular ions, i.e., that the proton permeability of the cell membrane has markedly increased. Notably, the increase in proton permeability requires the presence of chloride or bromide ions: the pH recovery was not observed in nitrate-containing medium (Fig. 2D).
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reagent N-phenyl maleimide (NPM), which is essentially impermeant and, therefore, only oxidizes extracellularly facing-SH groups, completely inhibited the copper-induced increase in chloride permeability, the flux being 44.4 6 4.2 and 42.8 6 0.73 mmol/L cells/h in copper-treated and control cells, respectively. When NPM-pretreated cells were exposed to 100 mM CuSO 4 in the presence of 1 mM ascorbate, NPM failed to
FIG. 2. Effect of 50 mM CuSO 4 on the permeability of the erythrocyte membrane to proton equivalents. pH o recovery after acidification of weakly buffered media (1 mM HEPES–KOH) of different anion composition was monitored using Beckman pH electrode. Data shown are from representative experiments. (A) Lack of pH o recovery after addition of 0.1 M HCl aliquot into copper-free lamprey erythrocyte suspension, n 5 4. (B–D) pH o recovery after acidification caused by hydrolysis of CuSO 4 added to poorly buffered media. (B) pH o recovery in the standard Cl 2-containing medium, n 5 5. (C) pH o recovery in Br 2-containing medium, n 5 4. (D) pH o recovery in NO 32containing medium, n 5 5. Filled symbols give pH o values in copper-free red cell suspensions, and empty symbols give the values in copper-containing suspensions.
Effect of Copper Ions on Cl 2 Influx Exposure of lamprey erythrocytes to 100 mM copper sulfate caused a pronounced increase in the uptake of Cl 2 in the erythrocytes after an approximately 3-min lag period (Fig. 3A). The maximal rate of Cl 2 influx, 207 6 31 mmol/L cells/h (as compared to 55.8 6 3.2 mmol/L cells/h in control), was measured after a 5-min copper treatment. The result indicates that not only the proton permeability but also the chloride permeability of the red cell membrane is markedly increased by copper. Chloride influx was further increased to 802 6 93 mol/L cells/h already during the first 3–5 min of treatment, when the copper exposure was carried out in the presence of a reducing agent, 1 mmol ascorbate (Fig 3B). Notably, ascorbate alone did not influence the chloride flux, which was 53.7 6 4.3 mmol/L cells/h in ascorbate-treated cells. In contrast, pretreatment of the cells with the sulphydryl
FIG. 3. Effect of CuSO 4 on Cl 2 influx in lamprey erythrocytes. (A) Time course of 36Cl uptake under control conditions (E) in the presence 100 mM CuSO 4 (■) and in the presence of 1 mM ascorbate and 100 mM CuSO 4 (h). Values are means for 5–12 independent experiments 6 SEM. (B) Effect of CuSO 4 on unidirectional Cl 2 influx in the presence of 1 mM ascorbate to 1 mM NPM in the incubation medium. Unidirectional Cl 2 influx was calculated from 10 min 36Cl uptake for control (open bars) and for 2 min tracer uptake in copper-treated cells (hatched bars). Before the tracer was added, the cells were preincubated for 5 min with 100 mM CuSO 4 or for 2 min 1 mM ascorbate 1 100 mM CuSO 4. When 1 mM NPM was used, the cells were pretreated for 15 min before copper were added and then incubated for 10 more min with 100 mM CuSO 4. Asterisks denote the statistical significance of the difference between the means for copper-treated and control cells: ***p , 0.001. Values are means of 5 independent experiments 6 SEM.
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FIG. 5. Effect of CuSO 4 on the erythrocyte water content as a function of time at 5, 10, 15, or 20 min incubation with (■) or without (h) 100 mM CuSO 4. Asterisks denote statistical significance of the difference in mean of the control and copper-treated cells at the same time point, ***p , 0.001. Experiments are means of 6 independent experiments 6 SEM.
copper-sensitive Cl 2 influx already at 10 mM CuSO 4 concentration (at which concentration copper did not affect chloride flux in 155 mM chloride medium; Fig. 4). Effect of Copper Ions on Cell Water and Cation Fluxes FIG. 4. Effect of CuSO 4 on 36Cl uptake in the media of different anion composition. Incubation media contained (mM) NaCl/NaBr/NaNO 3, 140; HEPES–KOH, 5; CaCl 2, 1; glucose, 10. 36Cl uptake was measured during 10 min incubation with the tracer in the absence of copper (open bars), or in the presence of 10 mM CuSO 4 (hatched bars) or 100 mM CuSO 4 (cross-hatched bars). Values are means of 5 independent experiments 6 SEM. ***p , 0.0001 as compared to control.
prevent copper-induced activation of Cl 2 influx (data not shown). All the above data, the simultaneous increase in both proton and chloride transport rates, the effect of reducing agent, and the effect of extracellular sulphydryl modification suggest that cupric ions are reduced to cuprous ions on the extracellular membrane surface, and that these cuprous ions then induce an electroneutral Cl 2/OH 2 exchange across the cell membrane, as has been observed in asolectin and renal brush border membrane vesicles (Karniski, 1992). In asolectin vesicles (Karniski, 1992) replacement of external Cl 2 by Br 2 facilitates Cu 1mediated chloride/hydroxyl exchange. In nitrate-containing medium cuprous ions get spontaneously oxidized back to cupric ions, so no anion exchange should be observed. The effect of anion substitution on the effect of copper on chloride flux was, therefore, tested. As can be seen from Fig. 4, reducing the extracellular chloride concentration to 2 mM and replacing it by nitrate abolished the activation of 36Cl uptake in the presence of 100 mM CuSO 4. In contrast, similar substitution of chloride by bromide resulted in a pronounced activation of the
Exposure of lamprey erythrocytes to 100 mM CuSO 4 results in a significant increase in cellular water content, already within 15 min of exposure (Fig. 5). Copper-induced swelling is associated with dose-dependent increase in cellular Na 1 and TABLE 1 Effect of CuSO 4 on the Lamprey Erythrocyte Na 1 and K 1 Content Intracellular cation concentration, (mmol/L cells) Treatment
Na1
K1
Control 25 mM CuSO 4 50 mM CuSO 4 100 mM CuSO 4 Amiloride, 1 mM 100 mM CuSO 4 1 amiloride Ba 21, 1 mM 100 mM CuSO 4 1 Ba 21
31.3 6 1.6 35.2 6 2.4 37.6 6 1.8* 63.4 6 8.5*** 30.3 6 3.3 33.3 6 4.0 33.5 6 2.4 47.9 6 7.6***
63.1 6 7.1 58.0 6 7.0* 50.4 6 6.1** 44.6 6 6.8*** 63.4 6 6.1 59.6 6 5.2 64.4 6 4.6 46.7 6 8.9***
Note. Cell suspension was treated with 100 mM CuSO 4 for 20 min. Cells were separated by centrifugation and washed free from external Na 1 and K 1 with Mg 21-sucrose solution. The cell pellet was lysed, and cell K 1 and Na 1 content were assayed by flame photometry. Values of 5 independent experiments are means 6 SEM. * p , 0.05. ** p , 0.01. *** p , 0.001 value vs control.
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TABLE 2 Effects of CuSO 4, Ba 21, and Amiloride on Unidirectional K 1 Influx into the Lamprey Erythrocytes K 1 influx, (mmol/L cells/h) Additions
Control
100 mM CuSO 4
None 1 mM Ba 21 1 mM amiloride
1.70 6 0.26 1.06 6 0.11*** 0.99 6 0.12***
1.90 6 0.25 1.67 6 0.26 ### 1.01 6 0.09
Note. The cells were pretreated with the inhibitors for 15 min and then K 1( 86Rb) influx was measured. The presented values 6 SEM are average means of 7 independent experiments. *** A statistically significant difference to the control flux (p , 0.001). ### Significant difference (p , 0.001) as compared to the flux in Ba 21-treated cells in the absence of copper.
decrease in K 1 content (Table 1). Net Na 1 uptake and K 1 loss by the cells exposed to 100 mM CuSO 4 for 20 min is abolished by pretreatment of the cells with 1 mM amiloride, but not with 1 mM Ba 21. To characterize the fluxes involved, we measured both the conductive cation fluxes using the whole cell patch clamp technique and radioactive tracer fluxes in the presence and absence of several ion transport inhibitors. It was previously shown that under physiological conditions the major charge-carrying pathway in lamprey erythrocyte membrane is K 1-selective, with very little current carried by Na 1 or Cl 2 ions (Virkki and Nikinmaa, 1996, 1998). Figure 6 shows a current-voltage relationship as recorded with KCl solution in the pipette and KCl or NaCl media in the bath in the absence or presence of 100 mM CuSO 4. The whole-cell con-
FIG. 6. Effect of 100 mM CuSO 4 on the lamprey erythrocyte membrane conductance to Na 1 and K 1. Whole-cell patch clamp technique was used with KCl solution in the pipette and either NaCl or KCl solutions in the bath. The figure shows representative data of seven experiments.
FIG. 7. Time course of 22Na uptake in lamprey erythrocytes exposed to 100 mM CuSO 4. Copper was added to the cell suspension together with the radioactive tracer. 22Na uptake by copper-treated cells differs significantly ( p , 0.001) from controls already after 3 min of incubation. Values are means 6 SEM of 6 independent experiments.
ductance shows strong inward rectification in symmetric potassium-containing solutions, as observed previously (Virkki and Nikinmaa, 1996). Replacement of K 1 by Na 1 in the bath results in a strong decrease in inward conductance. However, addition of cupric sulphate (25, 50, or 100 mM; n 5 2, 3, and 7, respectively) to the bath solution does not affect either inward or outward whole-cell currents in either sodium- or potassium-containing media. Thus, the possibility of copperinduced activation of any conductive pathway for K 1, Na 1, or Cl 2 can be ruled out. Using sodium-22, we measured the effect of copper on the sodium fluxes. There was a marked increase in both unidirectional influx and efflux of sodium across lamprey erythrocyte membrane after an initial 3–5 min lag period (Fig. 7). In the presence of 100 mM CuSO 4 the rate coefficient for influx was maximally 6.1 6 0.5 h 21, as compared to 0.15 6 0.02 h 21 in control conditions. The rate coefficients for efflux were 11.7 6 1.4 h 21 for copper-exposed cells and 0.89 6 0.18 h 21 in controls. Copper-induced Na 1 efflux was completely abolished by pretreatment with 1 mM amiloride. Dose-response of MIA effect on Na 1 influx in copper-free media gave IC 50 of 2.45 6 0.45 mM with the flux observed after maximal inhibition of 0.075 h 21. However, dramatic increase in Na 1 influx induced after 10 min treatment by 100 mM CuSO 4 was only inhibited by approximately 70% by 1 mM amiloride or 100 mM MIA (Fig. 8). Hence, in addition to stimulation of sodium influx via the sodium/proton exchanger, copper treatment results in activation of the amiloride (MIA)insensitive Na 1 transport pathway. Most likely, the same amiloride-insensitive component of Na 1 influx is preserved when the NPM-pretreated cells are exposed for 10 min to 100 mM CuSO 4. After 15 min pretreatment with 1 mM NPM, Na 1 influx in the presence of copper was 5.01 6 0.11 h 21 whereas
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With regard to potassium transport, exposure to copper resulted in a net potassium loss from the cell. However, the effect of copper ions on the inhibitor sensitivity of potassium fluxes in lamprey erythrocytes depends on the copper concentration used. The potassium efflux induced at 25 mM CuSO 4 was only slightly sensitive to amiloride, but completely inhibited by 1 mM Ba 21 (Fig. 9). In contrast, the potassium efflux induced at 100 mM CuSO 4, was hardly affected by Ba 21 but effectively inhibited by amiloride. Although copper exposure affected the net loss of potassium from the cells markedly, it did not influence the influx of potassium (as tested by measuring the unidirectional 86Rb influx) at 100 mM CuSO 4 (Table 2) but prevented Ba 21-induced inhibition of K 1 influx. DISCUSSION
FIG. 8. Effect of the inhibitors of Na 1/H 1 exchanger on copper-activated Na 1 influx. The cells were preincubated for 15 min with either MIA (10 or 100 mM) or amiloride (1 mM) before the addition of 100 mM CuSO 4. Radioactive tracer was added 5 min after the toxicant and uptake followed for 2 min. Flux values in copper-treated cells differ significantly ( p , 0.001) from controls regardless of inhibitors’ application. Values are means of 5 experiments 6 SEM.
in copper-free NPM-pretreated cells it was 0.156 6 0.016 h 21. After replacement of external Cl 2 by NO 32, 10 min treatment with 100 mM CuSO 4 resulted in increase in Na 1 influx from 0.198 6 0.028 h 21 to 1.79 6 0.12 h 21 compared to 0.156 6 0.008 h 21 vs 9.09 6 0.23 h 21 in the chloride-containing medium without and with CuSO 4, respectively. In bromide-containing saline 100 mM CuSO 4 increased Na 1 influx from 0.116 6 0.0037 to 13.5 6 0.36 h 21. Thus, replacement of extracellular Cl 2 by nitrate, preventing copper-induced increase in proton permeability, reduced the sodium influx to the same level as inhibition of the sodium/proton exchanger by MIA. It indicates that the copper-induced increase in sodium/ proton exchange activity is most likely secondary to the changes in intracellular pH. Moreover, an unidentified Na 1 transport pathway is induced by cupric ions in parallel to Na 1/H 1 exchange activation, since NPM pretreatment is not abolishing its activation by CuSO 4.
We used high bulk concentrations of copper sulfate to induce membrane level disturbances in lamprey erythrocytes rapidly. Actual concentration of free cupric ions in the medium was not measured but did not exceed 10 210 M due to high affinity of Cu 21 ions to such ligands as chloride, hydroxyl, and Tris ions, as follows from complex stability constants given by Smith and Martell (1976). The observed disturbances, cell swelling, net ion losses, and pH disturbances are similar to those observed in Ehrlich ascites tumor cells treated with CuSO 4 (Kramhoft et al., 1988; Lambert et al., 1984). Thus, at the membrane level, it is possible that the mechanisms implicated for lamprey erythrocytes are important behind the toxic effects in other cell types and also in vivo. Two observations suggest that, initially, the divalent form of
FIG. 9. Net K 1 loss into K 1-free incubation medium from the lamprey erythrocytes exposed to either 25 or 100 mM CuSO 4 for 20 min. 1 mM BaCl 2 (hatched bars) or 1 mM amiloride (cross-hatched bars) was added to the cell suspension 15 min prior to copper sulphate. Open bars represent K 1 loss from the copper-treated cells in the absence of inhibitors. Asterisks denote statistical difference (*p , 0.05 and ***p , 0.001) of K 1 loss as compared to potassium loss from the cells into copper-free medium (dashed line). Values are means of 6 independent experiments 6 SEM.
COPPER EFFECTS ON ION TRANSPORT
copper is reduced to a univalent form which causes the membrane level effects. First, the initial lag period in the copper effect on ion fluxes disappears in the presence of the reducing agent, ascorbate, and, second, ascorbate accentuates the effects of copper on the measured parameters. Extracellularly facing sulphydryl groups appear to be involved in the reduction of divalent copper to monovalent copper, because their oxidation to –S–S– bonds fully inhibits the copper-induced activation of Cl 2 influx. Notably, reduction of cupric ions to cuprous ions is an important step of copper uptake in a number of cell types including mammalian hepatocytes, C-6 and K562 cell lines, and in yeast cells (Qian et al., 1995; Davidson et al., 1994; Hasset and Kosman, 1995). In the plasma membrane of rat hepatocytes the electron transfer required for the formation of cupric ions is catalyzed by NADH oxidase (Van den Berg and McArdle, 1994), an enzyme which is likely to be present also on lamprey red cell membrane. In the yeast Saccharomyces cerevisiae, cell surface cupric reductases Fre1p and Fre2p are involved in the facilitation of copper transport across the membrane (Georgatsou et al., 1997). Cuprous ions form relatively poorly water-soluble, and thus lipophilic, ion pairs with halides (chloride and bromide) and hydroxyl ion. These ion pairs incorporate into the lipid bilayer and mediate electroneutral halide/hydroxyl ion exchange, thus increasing the apparent halide and proton (or actually hydroxyl ion) permeability. This conclusion is supported by the observation that the chloride and proton (hydroxyl ion) permeabilities are reduced with increasing water solubility (decreasing lipid solubility) of the ion pair: the effect is greater with bromide with a solubility constant of 4.15p10 28 for CuBr than with chloride with a solubility constant of 1.02p10 26 mol/L for CuCl. Since Cu 1 is unstable in nitrate-containing medium, ion pair formation does not occur and, thus, no changes in anion (or apparent proton) permeability take place. Univalent copper ions also induce electroneutral chloride/hydroxyl ion exchange across model membranes and across renal brush border membrane vesicles (Karniski, 1992). Halide/hydroxyl ion exchange across plasma membranes and lipid bilayers via similar mechanism is also induced by tributyltin, Hg 21, and Tl 31 (Gutknecht, 1981; Wieth and Tosteson, 1979; Karniski, 1992; Dias and Monreal, 1994). In addition to the mechanism presented above, the copperinduced increase in proton permeability could be caused by primary activation of the sodium/proton exchange, the major proton transporting pathway of lamprey erythrocytes (Virkki and Nikinmaa, 1994), as has been described for Ehrlich ascites tumor cells (Kramhoft et al., 1988). However, this is not the case for lamprey erythrocytes. With the proton and sodium gradients present across lamprey erythrocyte membrane (Virkki and Nikinmaa, 1994), activation of the sodium/proton exchange by copper would have resulted in intracellular alkalinization instead of the pronounced acidification observed. Furthermore, the simultaneous increase in both the intracellular
211
proton and the sodium activity is incompatible with direct effects of copper on the sodium/proton exchanger. On the other hand, the copper-induced increase in the sodium permeability appears to be largely due to a secondary activation of the sodium/proton exchange as a consequence of the initial cellular acidification which is a powerful stimulus for the activation of the sodium/proton exchange in lamprey erythrocytes (Virkki and Nikinmaa, 1994). The increase in sodium influx is markedly inhibited by MIA, and the amiloride-sensitive sodium flux is much less increased by copper treatment in nitrate-containing medium (in which acidification is not observed) than in chloride containing medium. A minor proportion of the copper-induced increase in sodium permeability is due to an amiloride-insensitive sodium transport pathway which does not require intracellular acidification for activation and is sensitive to extracellular divalent copper ions. In molluscan neurons 1–100 mM Cu 21, 100 mM Hg 21, or Ag 1 induced rapid irreversible depolarization accompanied by an increase in membrane conductance for Na 1 (Weinreich and Wonderlich, 1987). However, based on our patch-clamp data, it is unlikely that the transport pathway activated in lamprey would be a channel, since the ion conductivity is not affected by copper treatment. With regard to potassium transport, copper did not affect membrane premeability. However, there was a net potassium loss from copper-exposed erythrocytes which at low copper concentrations (25–50 mM) probably occurred via the Ba 21sensitive, low conductance K 1 channel (Virkki and Nikinmaa, 1996), and at the higher copper concentration (100 mM) via the Ba 21-insensitive, swelling-activated channel (Virkki and Nikinmaa, 1998). Inhibitory action of Cu 21 on Na 1/K 1 pump has been reported for many cell types (e.g., Li et al., 1996). However, since copper ions did not affect K 1 influx, Na 1/K 1 ATPase was not inhibited in lamprey erythrocytes in the present experiments. Notably, the effects described in the present study are mainly caused by extracellular copper ions. The inhibition of Na 1/K 1 ATPase, on the other hand, requires that Cu 21 ions prevent interaction of the enzyme with Mg 21 in the cytosolic side of the membrane (Li et al., 1996). We have not investigated rates of copper transport into the lamprey erythrocyte. However, low transport rates for copper ions can be expected, since in erythrocytes copper transport normally occurs mainly via the anion exchange pathway (Alda and Garay, 1988), and lamprey erythrocytes are characterized by a virtual absence of anion exchange (e.g., Nikinmaa and Railo, 1987; Tufts and Boutilier, 1989). In conclusion, the membrane effects of copper ions appear to involve the following sequence of events. (1) Cu 21 is reduced to Cu 1 with integral protein having essential externally facing SH-groups catalyzing the reaction. (2) Consequently, poorly water-soluble electroneutral ion pairs CuCl or CuOH incorporate into the lipid bilayer and shuttle between the internal and external surface of the membrane inducing a rapid Cl 2/OH 2
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exchange. (3) Protonophore-like action of Cu 1 results in dissipation of the transmembrane proton gradient and marked intracellular acidification. (4) Decrease in pH i activates the amiloride-sensitive Na 1/H 1 exchange. (5) The net uptake of chloride and sodium cause the cell swelling, and, consequently, net potassium efflux occurs mainly via the swelling-activated pathways. In addition, interaction of cupric ions with the membrane activates an amiloride-insensitive Na 1 transport pathway of unknown nature. The data of the present study and the proposed mechanisms for copper-induced effects are compatible with earlier results on the toxic effects of copper, i.e., osmolarity disorders (Heath, 1997), net loss of ions and acidosis observed in gills (Wilson and Taylor, 1993), collapse of transmembrane proton gradient on mitochondrial membrane after penetration of copper ions into the cells (Wojczak et al., 1996), and net K 1 loss from copper-treated yeast cells (Passow and Rothstein, 1960). The interesting parallel between the effects of copper and the effects of organic tin chlorides suggest that the wide use of copper salts in agriculture as fungicide, as a food preservative, and for textile dyeing and metal coloring should be carefully monitored with regard to the toxic effects. ACKNOWLEDGMENTS
Gusev, G. P., and Sherstobitov, A. O. (1996). An amiloride-sensitive, volumedependent Na 1 transport across the lamprey (Lampetra fluviatilis) erythrocyte membrane. Gen. Physiol. Biophys. 15, 129 –143. Gutknecht, J. (1981). Inorganic mercury (Hg 21) transport through lipid bilayer membrane, J. Membr. Biol. 61, 61– 66. Gwozdzinski, K. (1991). A spin label study of the action of cupric and mercuric ions on human red blood cells. Toxicology 65, 315–323. Hansen, H. J. M., Olsen, A. G., and Rosenkilde, P. (1996). The effect of Cu on gill and esophagus lipid metabolism in the rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 113C, 23–29. Harris, E., and Giltin, J. D. (1996). Genetic and molecular basis for copper toxicity. Am. J. Clin. Nutr. 63, 836S– 841S. Hassett, R., and Kosman, D. J. (1995). Evidence for Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae. J. Biol. Chem. 270, 128 –134. Heath, A. G. (1997). Water Pollution and Fish Physiology. CRC Press, Boca Raton, FL. Karniski, L. P. (1992). Hg 21 and Cu 1 are ionophores, mediating Cl 2/OH 2 exchange in liposomes and rabbit renal brush border membranes. J. Biol. Chem. 267, 19218 –19225. Kramhoft, B., Lambert, I. H., and Hoffmann, E. K. (1988). Na 1/H 1 exchange in Ehrlich ascites tumour cells: Activation by cytoplasmic acidification and by treatment with cupric sulphate. J. Membr. Biol. 102, 35– 48. Lambert, I. H., Kramhoft, B., and Hoffmann, E. K. (1984). Effect of copper on volume regulation in Ehlich ascites tumour cells. Mol. Physiol. 6, 83–98. Li, J., Lock, R. A. C., Klaren, P. H. M., Swarts, H. G. P., Schuurmans Stekhoven, F. M. A. H., Wendelaar Bonga, S. E., and Flik, G. (1996). Kinetics of Cu 21 inhibition of Na 1/K 1 ATPase, Toxicol. Lett. 87, 31–38.
This work was supported by Academy of Finland, Research Council for the Environment and Natural Resources (grant 40830) for M.N., and CIMO scholarship and Hali-Gali Club scholarship for A. B. Riitta Niemi is acknowledged for excellent technical assistance and A. A. Lew and A. O. Sherstobitov for valuable discussions.
Nikinmaa, M., and Huestis, W. H. (1984). Adrenergic swelling in nucleated erythrocytes: cellular mechanisms in a bird, domestic goose and two teleosts, striped bass and rainbow trout. J. Exp. Biol. 113, 215–224.
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