Lars Ole Simonsen

Volume-induced increase of K+ and Cl- permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+.

SummaryEhrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl−, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca2+-activated K+ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca2+ ionophore A23187 in the presence of external Ca2+, and also by a sudden increase in external Ca2+. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca2+ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl− permeability of the cell membrane there was no change in cell volume or Cl− content when the K+ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca2+ ionophore A23187 in isotonic medium promoted Cl− loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl− permeability was increased in parallel with and even more than the K+ permeability. It is proposed that separate conductive K+ and Cl− channels are activated during regulatory volume decrease by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin.

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.

Separate, Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

SummaryThe net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl− transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl− transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl− conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl− conductance is rate limiting. An A23187-induced activation of42K and36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca2+-activated K+ channel. (ii) unaffected by substitution of NO3− or SCN− for Cl−, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl− conductance. The Cl− conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl− transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl− transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl− transport pathway. The activated anion transport pathway is unselective, carrying both Cl−, Br−, NO3−, and SCN−. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl− transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by42K flux experiments and indirectly in media where the dominating anion (SCN−) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from42K flux measurements at high external K+, and from net K− flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

SummaryEhrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. 1. The volume recovery was inhibited when NO3− was substituted for Cl−, and when Na+ was replaced by K+, or by choline (at 5mm external K+). 2. The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. 3. The net uptake of Cl− was much larger than the value predicted from the conductive Cl− permeability. The unidirectional36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. 4. During volume recovery the Cl− flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl−, or with an uptake via a K+, Na+, 2Cl− cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. 5. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl− in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+, Cl− cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+, Cl− cotransport system.

The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation.

SummaryIntracellular potentials were measured, using a piezoelectric electromechanical transducer to impale Ehrlich ascites tumor cells with capillary microelectrodes. In sodium Ringers, the potential immediately after the penetration was −24±7 mV, and decayed to a stable value of about −8 mV within a few msec. The peak potentials disappeared in potassium Ringers and reappeared immediately after resuspension in sodium. Ringers, whereas the stable potentials were only slightly influenced by the change of medium. The peak potential is in good agreement with the Nernst potential for chloride. This is also the case when cell sodium and potassium have been changed by addition of ouabain. It is concluded that the peak potentials represent the membrane potential of the unperturbed cell, and that chloride is in electrochemical equilibrium across the cell membrane.The membrane potential of about −11 mV previously reported corresponds to the stable potential in this study, and is considered as a junction potential between damaged cells and their environment. Similar potential differences were recorded between a homogenate of cells and Ringers.The apparent membrane resistance of Ehrlich cells was about 70 Ωcm2. This is two orders of magnitude less than the value calculated from36Cl fluxes, and may, in part, represent a leak in the cell membrane.For comparison, the influence of an eventual leak on measurements in red cells and mitochondria is discussed.

Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells.

1. The steady‐state tracer exchange flux of chloride was measured at 10‐150 mM external chloride concentration, substituting either lactate or sucrose for chloride. The chloride flux saturates in both cases with a K 1/2 about 50 and 15 mM, respectively. 2. The inhibitory effect of other monovalent anions on the chloride transport was investigated by measuring the 36Cl‐ efflux into media where either bromide, nitrate, or thiocyanate had been substituted for part of the chloride. The sequence of increasing affinity for the chloride transport system was found to be: Br‐ less than Cl‐ less than SCN‐ = NO3‐. 3. The chloride steady‐state exchange flux in the presence of nitrate can be described by Michaelis‐Menten kinetics with nitrate as a competitive inhibitor of the chloride flux. 4. The apparent activation energy (EA) was determined to be 67 +/‐ 6.2 kJ/mole, and was constant between 7 and 38 degrees C. 5. The membrane potential (Vm) was measured as a function of the concentration of external K+, substituting K+ for Na+. The transference number of K+ (tK) was estimated from the slope of Vm vs. log10 (K+)e, and tCl and tNa were calculated, neglecting current carried by ions other than Cl‐, K+, and Na+. The diffusional net flux of K+ was calculated from the steady‐state exchange flux of 42K+, assuming the flux ratio equation to be valid. From this value the K+ conductance and the Na+ and Cl‐ conductances were calculated. The experiments showed that GCl, GNa, and GK are all about 14 muS/cm2. 6. The net (conductive) chloride permeability derived from the chloride conductance was 4 x 10(‐8) cm/sec compared with the apparent permeability of 6 x 10(‐7) cm/sec as calculated from the chloride tracer exchange flux. These data suggest that about 95% of the chloride transport is mediated by an electrically silent exchange diffusion. 7. Comparable effects of phloretin (0.25 mM) on the net (conductive) permeability and the apparent permeability to chloride (about 80% inhibition) may indicate that the chloride exchange and conductance pathways are not completely separate and distinct modes of transport, but may involve common elements. The reduced chloride permeability in the presence of phloretin is estimated to be two orders of magnitude larger than the ground permeability of the cell membrane.

On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells

Abstract. The putative role for Ca2+ entry and Ca2+ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P3 content, as measured in extracts from [2-3H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca2+-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca2+-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca2+ (1% only at 0.1-mm Ca2+ and in Ca2+-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca2+ upon the volume-induced K+ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca2+ chelator BAPTA when the BAPTA-loading is performed in K+ equilibrium medium; (vi) an inhibition of the swelling-induced K+ loss (about 50%) at 1-mm Ba2+, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 μm), reported inhibitors of the K+ loss induced by Ca2+-mobilizing agonists. Thus, Ca2+signaling by Ca2+ release or Ca2+ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells.

Cell Volume Regulation: Intracellular Transmission

The cell membranes of virtually all animal cells are highly permeable to water, and hence the cell volume will be determined by the cellular content of osmotically active solutes and by the osmolality of the extracellular fluid. It should be noted, however, that in epithelial cells only the membrane on the basolateral side is highly water-permeable. The apical membrane can be quite impermeable to water, as e.g. in the epithelial cells of late distal tubules and collecting ducts of the kidney in the absence of antidiuretic hormone.

Mechanisms in Volume Regulation in Ehrlich Ascites Tumor Cells

The Ehrlich ascites tumor cell has been used as a model of an unspecialized mammalian cell, in an attempt to disclose the mechanisms involved in the regulation of cellular water and salt content. In hypotonic medium Ehrlich cells initially swell as nearly perfect osmometers, but subsequently recover their volume within about 10 min with an associated net loss of KCl, amino acids, taurine and cell water. The net loss of KCl takes place mainly via separate, conductive K+ and Cl- transport pathways, and the net loss of taurine through a passive leak pathway. Ca2+ and calmodulin appear to be involved in the activation of the K+ and Cl- channels, as well as the taurine leak pathway. In hypertonic medium Ehrlich cells initially shrink as osmometers, but subsequently recover their volume with an associated net uptake of KCl and water. In this case, the net uptake of KCl is the result of the activation of an electroneutral, Na+- and Cl- -dependent cotransport system with subsequent replacement of cellular Na+ by extracellular K+ via the Na+/K+ pump. In the present review we describe the ion and taurine transporting systems which have been identified in the plasma membrane of the Ehrlich ascites tumor cell. We have emphasized the selectivity of these transport pathways and their activation mechanisms. Finally, we propose a model for the activation of the conductive K+ and Cl- transport pathways in Ehrlich cells which includes Ca2+, leukotrienes, and inositol phosphate as intracellular second messengers.

Exchangeability of chloride in ehrlich ascites tumor cells.

Abstract In Ehrlich cells 30–50% of cell Cl − has been reported to be non-exchangeable. In the present study the possibility of protein interference with the Cl − titration was eliminated by deproteinization with HClO 4 , or with ZnSO 4 -NaOH followed by perborate oxidation, or by alkaline dry ashing. Cell Cl − is demonstrated to be completely exchangeable with 36 Cl − and with NO 3 − , and there is no evidence of compartmentation. However, protein interference with the argentimetric titration may introduce substantial error, mimicking a fraction of non-exchangeable cell Cl − . For cells equilibrated at 38° in sodium Ringer solution with a Cl − concentration of 151 mM, the Cl − concentration was 58 μmole/ml cell water, and this value is consistent with a passive distribution of Cl − .