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.     

Role of prostaglandins and leukotrienes in volume regulation by Ehrlich ascites tumor cells

Summary PGE₂ and LTC₄ syntheses in Ehrlich ascites cells were measured by radioimmunoassay. Hypotonic swelling results in stimulation of the leukotriene synthesis and a concomitant reduction in the prostaglandin synthesis. If the cells have access to sufficient arachidonic acid there is a parallel increase in the synthesis of both leukotrienes and prostaglandins following hypotonic exposure. PGE₂ significantly inhibits regulatory volume decrease (RVD) following hypotonic swelling in Na-containing medium but not in Na-free media, supporting the hypothesis that the effect of PGE₂ is on the Na permeability. PGE₂ also had no effect on RVD in Na-free media in the presence of the cation ionophore gramicidin. Since the Cl permeability becomes rate limiting for RVD in the presence of gramicidin, whereas the K permeability is rate limiting in its absence, it is concluded that PGE₂ neither affects Cl nor K permeability. Addition of LTD₄ accelerates RVD and since the K permeability is rate limiting for RVD this shows that LTD₄ stimulates the K permeability. Inhibition of the leukotriene synthesis by nordihydroguaiaretic acid inhibits RVD even when a high K conductance has been ensured by the presence of gramicidin. It is, therefore, proposed that an increase in leukotriene synthesis after hypotonic swelling is involved also in the activation of the Cl transport pathway.     

Relation between cytoskeleton,hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells

Summary Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca²⁺ or Ca²⁺-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca²⁺ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K⁺ ionophore valinomycin.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge

Summary Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na⁺/H⁺ exchanger, the Na/K pump, the Na⁺+K⁺+2Cl^– cotransporter and by loss of K⁺ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl^–] _i (100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na⁺, K⁺ and Cl^–, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl^– flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na⁺+K⁺+Cl^–], as well as the ratio of intra- to extracellular [Cl^–]. Both factors appear to influence the activity of the cotransport pathway.     

Effect of arachidonic acid,fatty acids,prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells

Summary Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE₂ inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE₂-induced Na uptake. PGE₂ has no effect on the volume-activated K and Cl permeabilities. LTB₄, LTC₄ and LTE₄ inhibit RVD insignificantly in hypotonically swollen cells. LTD₄, more-over, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD₄ even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.     

Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells

The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl^– but inhibited by addition of MK196 (anion channel blocker) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl^–, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a Mini Cl^– channel. The Cl^– efflux via this Cl^– channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS and inhibited by arachidonic acid and oleic acid. It is suggested that the swelling-activated Mini Cl^– channel and the swelling-activated taurine channel in the Ehrlich cell represent two distinct types of channels.This work was supported by the Danish Natural Research Council and by the NOVO foundation. Dr. Birte Kramhøft is acknowledged for critical reading of this paper.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

Self-inhibition of chloride transport in Ehrlich ascites tumor cells

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.     

Transmembrane ferricyanide reductase activity in Ehrlich ascites tumor cells

A transmembrane ferricyanide reductase activity was assayed in intact Ehrlich ascites tumor cells. Kinetic measurements gave a Km of 0.14 mM and a Vmax of 0.31 mumol/min per 10(6) cells. In short-term batch experiments, this activity was enhanced in the presence of 10 mM lactate, a source of cytosolic NADH. The transmembrane redox activity was accompanied by alkalinization of the cytosol. Both ferricyanide reduction and proton extrusion were diminished in the presence of 0.2 mM amiloride. Several cytotoxic drugs significantly inhibited the ferricyanide reductase activity at concentrations at which they show antineoplastic activity.