Poul Bennekou

Cobalt metabolism and toxicology--a brief update.

Cobalt metabolism and toxicology are summarized. The biological functions of cobalt are updated in the light of recent understanding of cobalt interference with the sensing in almost all animal cells of oxygen deficiency (hypoxia). Cobalt (Co(2+)) stabilizes the transcriptional activator hypoxia-inducible factor (HIF) and thus mimics hypoxia and stimulates erythropoietin (Epo) production, but probably also by the same mechanism induces a coordinated up-regulation of a number of adaptive responses to hypoxia, many with potential carcinogenic effects. This means on the other hand that cobalt (Co(2+)) also may have beneficial effects under conditions of tissue hypoxia, and possibly can represent an alternative to hypoxic preconditioning. Cobalt is acutely toxic in larger doses, and in mammalian in vitro test systems cobalt ions and cobalt metal are cytotoxic and induce apoptosis and at higher concentrations necrosis with inflammatory response. Cobalt metal and salts are also genotoxic, mainly caused by oxidative DNA damage by reactive oxygen species, perhaps combined with inhibition of DNA repair. Of note, the evidence for carcinogenicity of cobalt metal and cobalt sulfate is considered sufficient in experimental animals, but is as yet considered inadequate in humans. Interestingly, some of the toxic effects of cobalt (Co(2+)) have recently been proposed to be due to putative inhibition of Ca(2+) entry and Ca(2+)-signaling and competition with Ca(2+) for intracellular Ca(2+)-binding proteins. The tissue partitioning of cobalt (Co(2+)) and its time-dependence after administration of a single dose have been studied in man, but mainly in laboratory animals. Cobalt is accumulated primarily in liver, kidney, pancreas, and heart, with the relative content in skeleton and skeletal muscle increasing with time after cobalt administration. In man the renal excretion is initially rapid but decreasing over the first days, followed by a second, slow phase lasting several weeks, and with a significant long-term retention in tissues for several years. In serum cobalt (Co(2+)) binds to albumin, and the concentration of free, ionized Co(2+) is estimated at 5-12% of the total cobalt concentration. In human red cells the membrane transport pathway for cobalt (Co(2+)) uptake appears to be shared with calcium (Ca(2+)), but with the uptake being essentially irreversible as cobalt is effectively bound in the cytosol and is not itself extruded by the Ca-pump. It is tempting to speculate that this could perhaps also be the case in other animal cells. If this were actually the case, the tissue partitioning and biokinetics of cobalt in cells and tissues would be closely related to the uptake of calcium, with cobalt partitioning primarily into tissues with a high calcium turn-over, and with cobalt accumulation and retention in tissues with a slow turn-over of the cells. The occupational cobalt exposure, e.g. in cobalt processing plants and hard-metal industry is well known and has probably been somewhat reduced in more recent years due to improved work place hygiene. Of note, however, adverse reactions to heart and lung have recently been demonstrated following cobalt exposure near or slightly under the current occupational exposure limit. Over the last decades the use of cobalt-chromium hard-metal alloys in orthopedic joint replacements, in particular in metal-on-metal bearings in hip joint arthroplasty, has created an entirely new source of internal cobalt exposure. Corrosion and wear produce soluble metal ions and metal debris in the form of huge numbers of wear particles in nanometric size, with systemic dissemination through lymph and systemic vascular system. This may cause adverse local reactions in peri-prosthetic soft-tissues, and in addition systemic toxicity. Of note, the metal nanoparticles have been demonstrated to be clearly more toxic than larger, micrometer-sized particles, and this has made the concept of nanotoxicology a crucial, new discipline. As another new potential source of cobalt exposure, suspicion has been raised that cobalt salts may be misused by athletes as an attractive alternative to Epo doping for enhancing aerobic performance. The cobalt toxicity in vitro seems to reside mainly with ionized cobalt. It is tempting to speculate that ionized cobalt is also the primary toxic form for systemic toxicity in vivo. Under this assumption, the relevant parameter for risk assessment would be the time-averaged value for systemic cobalt ion exposure that from a theoretical point of view might be obtained by measuring the cobalt content in red cells, since their cobalt uptake reflects uptake only of free ionized cobalt (Co(2+)), and since the uptake during their 120 days life span is practically irreversible. This clearly calls for future clinical studies in exposed individuals with a systematic comparison of concurrent measurements of cobalt concentration in red cells and in serum.

The voltage-gated non-selective cation channel from human red cells is sensitive to acetylcholine

Using patch-clamp technique it is demonstrated, that the voltage-gated non-selective cation channel present in the human red cell is coupled to an acetylcholine receptor of nicotinic type. The concentration dependence of carbachol, the potency of selected agonists and an estimate of the numbers of channels/red cell are reported.

Differential effect of HOE642 on two separate monovalent cation transporters in the human red cell membrane.

Residual K⁺ fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K⁺ (Na⁺)/H⁺ exchanger as candidate pathways mediating this effect, although it is possible that these pathways represent different modes of operation of a single system. In the present study the effects of HOE642, recently characterised as an inhibitor of the K⁺(Na⁺)/H⁺ exchanger, on NSVDC has been determined to clarify this question. Radioisotope flux measurements and conductance determinations showed that HOE642 exerted differential effects on the NSVDC channel and the K⁺(Na⁺)/H⁺ exchanger, confirming that the salt loss observed in low ionic strength solutions represents contributions from at least two independent ion transport pathways. The findings are discussed in the context of red blood cell apoptosis (eryptosis) and disease.

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cells diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. Methodology/Principal Findings The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. Conclusions/Significance The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Calcium-induced oscillations in K+ conductance and membrane potential of human erythrocytes mediated by the ionophore A23187

The time-dependence of ionophore A23187-induced changes in the conductance of the Ca2+-sensitive K+ channels of the human red cell has been monitored with ion-specific electrodes. The membrane potential was reflected in CCCP-mediated pH changes in a buffer-free extracellular medium, and changes in extracellular K+ activity and electrode potential of an extracellular Ca2+-electrode were recorded. Within a narrow range of ionophore-mediated Ca2+ influx, the above-mentioned parameters were found to oscillate when ionophore was added to a suspension of glucose-fed cells. The period of oscillation was about 2 min/cycle depending on ionophore concentration, and the amplitude of hyperpolarization was about 60 mV, corresponding to a maximal gK+ of the same magnitude as gCl-. Without CCCP present no oscillation in K+ conductance was observed. The Ca2+ affinity for the opening process was in the micromolar range. The closing of the K+ channels was a spontaneous process in that the depolarization was well under way before the Ca2+-ATPase-mediated Ca2+ net efflux started. Below the Ca2+ influx range for oscillations, no response was observed for up to 20 min after the addition of ionophore. Above the upper limit, a permanent hyperpolarization resulted with an extracellular K+ activity increasing monotonically as a function of time. In experiments with ATP-depleted cells, responses of the latter type ensued at all ionophore concentrations above the lower limit. Addition of surplus EGTA to suspensions of hyperpolarized cells restores the normal membrane potential in the case of glucose-fed cells, whereas the K+-channels in ATP-depleted cells remained open.

K+-valinomycin and chloride conductance of the human red cell membrane: Influence of the membrane protonophore carbonylcyanide m-chlorophenylhydrazone

Chloride ion conductance of the human red cell membrane has been calculated, as the ratio between ion net charge flux and driving potential. The proton carrier CCCP was used to monitor changes in membrane potential following addition of valinomycin in sufficient quantities to raise the K+ conductance to a level comparable to the Cl- conductance. A K+-specific electrode was used to monitor changes in extracellular K+ concentration, and an H+-sensitive glass electrode for changes in extracellular pH, reflecting changes in membrane potential. The effects of varied concentrations of valinomycin and CCCP upon K+ and Cl- conductances were studied. It was found that, within an experimental error of about 10% S.D., the chloride conductance was constant for valinomycin concentrations in the range 1.0 X 10(-8)-1.0 X 10(-6), and for CCCP-concentrations in the range 2.0 X 10(-7)-2.0 X 10(-5) mol per litre cell suspension, while at a constant concentration of valinomycin the induced K+ conductance was considerably augmented by addition of CCCP.

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation.

Background Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. Design and Methods Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. Results In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO3-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na+/K+ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K+ uptake. Unidirectional K+ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K+,Cl− co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.

The human red cell voltage-regulated cation channel. The interplay with the chloride conductance, the Ca(2+)-activated K(+) channel and the Ca(2+) pump.

The activation/deactivation kinetics of the human erythrocyte voltage-dependent cation channel was characterized at the single-channel level using inside-out patches. It was found that the time dependence for voltage activation after steps to positive membrane potentials was slow (t1/2 about 30 s), whereas the deactivation was fast (t1/2 about 15 ms). Both activation and deactivation of this channel were also demonstrated in intact red cells in suspension. At very positive membrane potentials generated by suspension in extracellular low Cl− concentrations, the cation conductance switched on with a time constant of about 2 min. Deactivation of the cation channel was clearly demonstrated during transient activation of the Gárdos channel elicited by Ca2+ influx via the cation channel and ensuing efflux via the Ca2+ pump. Thus, the voltage-dependent cation channel, the Gárdos channel and the Ca2+ pump constitute a coupled feedback-regulated system that may become operative under physiological conditions.

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP.

SummaryThe ratio of valinomycin-mediated unidirectional K+ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K+ net efflux and42K influx. The driving force for the net efflux (Vm−EK+) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K+ net efflux has been calculated from the K+ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K+ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 μm and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.

The effect of ATP, intracellular calcium and the anion exchange inhibitor DIDS on conductive anion fluxes across the human red cell membrane

The influence of ATP depletion, the intracellular ionized Ca-concentration, anion substitution and DIDS on the conductive anion fluxes across the human red cell membrane has been examined. Under physiological or near physiological conditions it is not possible to observe conductive anion fluxes across the erythrocyte membrane in that anions totally dominate the membrane conductance. Consequently anions are at electro-chemical equilibrium and the netflux is zero. However, conductive anion fluxes can be induced by raising the potassium conductance, either by addition of valinomycin, or by triggering the native calcium activated potassium channel by addition of the Ca2+ ionophore A23187 to cells suspended in a calcium containing medium. The interpretation of data from experiments with valinomycin induced netfluxes has normally been done according to a constant field model, and the results have consequently been given as permeabilities. Since it has been demonstrated recently, that these cation pathways do not conform to a constant field scheme (Bennekou, P. and Christophersen, P. (1986) J. Membr. Biol. 93, 221-227 and Vestergaard-Bogind, B., Stampe, P. and Christophersen, P. (1985) J. Membr. Biol. 88, 67-75), it has been chosen, instead of permeabilities, to calculate the ion conductances from net efflux data, using an independent estimate of the membrane potential. The main result reported, is that only one component is found for the conductive anion fluxes in the presence of DIDS using the latter theoretical framework, whereas a sizeable DIDS-insensitive component is found when the constant field analysis is used. Furthermore it is found that ATP and intracellular calcium do not influence the anion conductances.