Permeabilization of Ehrlich ascites tumor cells by human serum

Human serum rapidly permeabilized Ehrlich ascites tumor cells to inorganic cations such as Rb⁺ and Ca²⁺; serum from several other species showed little or no activity. The effect of human serum was not reversed by washing the cells. Human serum, deficient in specific complement proteins, had no activity, but was reactivated by the addition of the missing complement component. Since Ca²⁺ was not required for the permeabilization, the alternative pathway of complement activation was implicated. Human serum deficient in Factor B of the alternative pathway was ineffective, but permeabilizing activity was restored by addition of Factor B. Rb⁺ uptake of several other cells was not inhibited by human serum. We conclude that an interaction between human complement and Ehrlich ascites tumor cells is responsible for the membrane lesion observed.     

Calcium-ion transport by intact Ehrlich ascites tumor cells at 0°C

At 0°C, where Ca²⁺ efflux is not observed, the uptake of Ca²⁺ by Ehrlich ascites tumor cells consists of four components: 1) An energy-dependent mitochondrial component, which is inhibited by uncouplers, respiratory inhibitors, and mitochondrial ATP-ase inhibitors. 2) Binding to the cell surface, which can be displaced by an EGTA wash. 3) An electrochemical gradient-dependent component, which is inhibited by agents which dissipate these gradients, such as proton ionophores, metabolic uncouplers, and valinomycin. The valinomycin inhibition of this transport component is dependent on K⁺ concentration. 4) Passive diffusion, which is dependent on Ca²⁺ concentration and is observed in the presence of inhibitors of the other components. The uptake of Ca²⁺ at 0°C is sensitive to ruthenium red presumably due to its competition with Ca²⁺ for cell binding sites.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

Effects of extremely-law-frequency electromagnetic fields on ion transport in several mammalian cells

We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca²⁺, Na⁺, K⁺, and H⁺) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), the intracellular pH (pH_i), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100–2000 μT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20–1200 μT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12–32% increase of the uptake of ⁴²K within a window at 14.5–15.5 Hz and 100–200 μT (rms), which was found in U937 and Ehrlich cells but not in the other cell types. © 1994 Wiley-Liss, Inc.     

KCl loss and cell shrinkage in the ehrlich ascites tumor cell induced by hypotonic media, 2-deoxyglucose and propranolol

Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca²⁺ ionophore, A23187, plus Ca²⁺ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO₃^? does not substitute for Cl^?; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl^? -Cl^? exchange, a net loss of K⁺ and a net gain of Na⁺ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca²⁺-activated, quinine-sensitive K⁺ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K⁺ through the Ca²⁺ stimulated pathway appears to be limited by Cl^? conductive movements; for when NO₃^?, an anion demonstrated here to have a higher conductive movement than Cl^?, is substituted for Cl^?, the cells will shrink when the Ca²⁺-stimulated K⁺ pathway is activated.     

Retention of calcium by mitochondria isolated from Ehrlich ascites tumor cells

Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells accumulate and retain high concentrations of Ca²⁺ in the presence of ATP for periods up to at least 20 min at 25 °C. The presence of inorganic phosphate up to 20 mm does not prevent such Ca²⁺ retention. The tumor mitochondria accumulate Ca²⁺ in the presence of succinate as an energy source but lose the Ca²⁺ after 1–2 min. Addition of ATP (K_m approx 1 mm) to the incubation medium after Ca²⁺ release, induces reaccumulation of the ion. Thus, the ability of the tumor mitochondria to retain Ca²⁺ differs markedly from that of rat liver mitochondria and is seen as being of potential biological significance to the unique metabolic behavior of the ascites tumor cells.     

Leukotriene-D4 induced cell shrinkage in Ehrlich ascites tumor cells

Summary The nature of the leukotriene-D₄ (LTD₄) induced cell shrinkage in Ehrlich ascites tumor cells has been investigated. LTD₄ treatment of Ehrlich cells induces net loss of cellular KCl and cell shrinkage independent of the initial cell volume. LTD₄ also produces water loss and reduction in cell volume when all extracellular and all intracellular Cl has been replaced by NO₃. On the other hand, LTD₄ fails to produce any significant changes in cell volume in the presence of the K-channel blocker quinine, suggesting that LTD₄ in Ehrlich cells induces Cl-independent K loss through the Ca²⁺-dependent K channels. However, the effect of physiological doses of LTD₄ on cell volume seems not to be as potent in Cl-free, NO₃ cells when compared to Cl-containing cells, indicating that LTD₄ in Ehrlich cells also provokes Cl-dependent K loss. LTD₄ seems not to produce K loss through an electroneutral K⁺/H⁺ exchange system. LTD₄ still produces Cl-independent K loss and cell shrinkage in the presence of the anticalmodulin drug pimozide but not in the presence of the LTD₄ receptor antagonist L-649,923 or the 5-lipoxygenase inhibitor NDGA. Pretreatment of the cells with pertussis toxin, which inactivates inhibitory guanine nucleotide binding proteins (G-proteins), leads to partial inhibition of the LTD₄-induced shrinkage. It is suggested that the LTD₄-induced activation of K and Cl transporting systems in Ehrlich ascites tumor cells is mediated via a G-protein coupled receptor and that LTD₄ might exert its effect through another lipoxygenase product. The Ca²⁺-calmodulin complex is not involved in the LTD₄-induced activation of K and Cl transporting systems.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Comparison of kinetic characteristics of Na+ -Ca2+ exchange in sarcolemma vesicles and cultured cells from chick heart

The kinetic characteristics of Na⁺ -Ca²⁺ exchange in isolated sarcolemma vesicles from new-borne chick heart, which contain about 70% of right-side-out vesicles, were compared with those of cultured embryonic chick heart cells. Na⁺ -Ca²⁺ exchange was monitored as Na_i-dependent Ca²⁺ uptake. Increase in the internal concentration of Na⁺ ([Na⁺]_i) in these two preparations caused increase in both the initial rate and the saturation-level of Ca²⁺ uptake. Plots of the rate of Ca²⁺ uptake against [Na⁺]_i showed similar saturation-kinetics in these two preparations. The apparent Michaelis constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) (0.35 mM) for Ca²⁺ uptake by the intact cells was much higher than that (0.031 mM) for Ca²⁺ uptake by the vesicles. The degree of inhibition by Mg²⁺ was also higher in the cells than in the vesicles. Some possible reasons (age of the chicks used, membrane potential, etc.), for these differences were examined and are discussed.     

Phosphate transport and its relationship to cation movements in Ehrlich Lettré ascites tumor cells.

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by ascites tumor cells. For this purpose, the incorporation of ³²P_i into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease ³²P_i uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 M_r anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases ³²P_i incorporation at concentrations as low as 2 × 10^?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na⁺ + K⁺)-ATPase and its dephosphorylation, stimulates the rate of incorporation of ³²P_i into cells and also raises the net inorganic phosphate level. The stimulation of ³²P_i incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K⁺, which is known to stimulate (Na⁺ + K⁺)-ATPase and its dephosphorylation, decreases ³²P_i incorporation. These observations suggest that anion transport and energy-dependent Na⁺ and K⁺ movements may be closely coupled to the intact cell.     

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.     

Characterization of calcium uptake into rough endoplamic reticulum of rat pancreas

Summary ATP-dependent Ca²⁺ uptake into isolated pancreatic acinar cells with permeabilized plasma membranes, as well as into isolated endoplasmic reticulum prepared from these cells, was measured using a Ca²⁺-specific electrode and⁴⁵Ca²⁺. Endoplasmic reticulum was purified on an isopycnic Percoll gradient and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the rough endoplasmic reticulum RNA was enriched threefold and the typical marker for the plasma membrane Na⁺,K⁺(Mg²⁺)ATPase was decreased 20-fold. When different fractions of the Percoll gradient were compared,⁴⁵Ca²⁺ uptake correlated with the RNA content and not with the Na⁺,K⁺(Mg²⁺)ATPase activity. The characteristics of nonmitochondrial Ca²⁺ uptake into leaky isolated cells and⁴⁵Ca²⁺ uptake into isolated endoplasmic reticulum were very similar: Calcium uptake was maximal at 0.3 and 0.2 mmol/liter free Mg²⁺, at 1 and 1 mmol/liter ATP, at pH 6.0 and 6.5, and free Ca²⁺ concentration of 2 and 2 mol/liter, respectively. Calcium uptake decreased at higher free Ca²⁺ concentration.⁴⁵Ca²⁺ uptake was dependent on monovalent cations (Rb⁺>K⁺>Na⁺>Li⁺>choline⁺) and different anions (Cl^–>Br^–>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–>SCN^–) in both preparations. Twenty mmol/liter oxalate enhanced⁴⁵Ca²⁺ uptake in permeabilized cells 10-fold and in vesicles of endoplasmic reticulum, fivefold. Calcium oxalate precipitates in the endoplasmic reticulum of both preparations could be demonstrated by electron microscopy. The nonmitochondrial Ca²⁺ pool in permeabilized cells characterized in this study has been previously shown to regulate the cytosolic free Ca²⁺ concentration to 0.4 mol/liter. Our results provide firm evidence that the endoplasmic reticulum plays an important role in the regulation of the cytosolic free Ca²⁺ concentration in pancreatic acinar cells.     

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

Summary Ehrlich 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.     

Pacemaking mechanism of the afterdischarge of the ovulation hormone-producing caudo-dorsal cells in the gastropod mollusc Lymnaea stagnalis

The ovulation hormone-producing caudo-dorsal cells (CDC) of Lymnaea stagnalis have three states of excitability (active, inhibited, and resting), which are related to the egg-laying cycle. Active state CDC produce a firing pattern of prolonged spiking activity (1 spike/2 s), which in the animal occurs shortly before egg laying. In preparations it is evoked as an afterdischarge upon repetitive stimulation of CDC. The afterdischarge is not synaptically driven, but rests on a pacemaking mechanism. CDC are silent in the inhibited and resting states, which follow egg laying. In these states the membrane potential is mainly dependent on [K⁺]₀. In the active state the ratio of the K⁺, Na⁺, and Ca²⁺ permeabilities has changed considerably, probably resulting from an increased permeability to Na⁺ and Ca²⁺. The firing rate in the afterdischarge is dependent on the membrane potential, which is confirmed experimentally by varying [K⁺]₀.[Na⁺]₀ and [Ca²⁺]₀ directly influence the firing rate. Firing stops in Na⁺-free saline, but is enhanced by Ca²⁺-free or high-Mg²⁺ saline. TTX does not affect firing. Relatively high concentrations of Co²⁺ and La³⁺ (2 × 10^?3M) strongly inhibit CDC. Regular firing can be changed into bursting by various means, such as high K⁺ or addition of 1 mM Ba²⁺. Bursting normally occurs at the beginning of the afterdischarge. Postburst hyperpolarizations are reduced in Ca²⁺-free saline and by low Co²⁺ (10^?4-5 10^?4M). Active CDC are driven by a pacemaking mechanism constituted by a voltage-dependent Na⁺/Ca²⁺ channel and a Ca²⁺-dependent K⁺ channel, thus resembling that of bursting pacemakers. The pacemaking mechanism is inactive in the resting and inhibited state.     

CALCIUM UPTAKE INTO RAT PHEOCHROMOCYTOMA CELLS1

Abstract– The uptake of ⁴⁵Ca²⁺ into cell suspensions prepared from a transplantable rat pheochromocytoma was measured. The uptake of Ca²⁺ into these cells is biphasic; there is a rapid, initial uptake of Ca⁺, followed by a slower uptake that proceeds at a linear rate for at least 10min at 37°C. The uptake of Ca²⁺ is a linear function of the external Ca²⁺ concentration over the range of 0.13-2.5 mm -Ca²⁺ Incubation of the cells in a medium containing 56mm -K⁺ results in a 2-3 fold increase in the uptake of Ca²⁺ into the cells; 56mm -K⁺ increases both phases of Ca²⁺ uptake. The cells apparently lack a mechanism to inactivate this 56 mm -K⁺-induced increase-in Ca²⁺ permeability. Two inhibitors of K⁺ stimulated catecholamine secretion, diphenylhydantoin and verapamil, both inhibit K ⁺-stimulated Ca²⁺ uptake. These results provide a direct demonstration of the stimulus-coupled uptake of Ca²⁺ into chromaffin cells, and provide additional evidence for the correlation of Ca²⁺ uptake with catecholamine secretion by these cells.     

Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture

Summary Ehrlich ascites tumor cells contain a Na⁺ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC₅₀ of 25 m and by dimethylamiloride with an IC₅₀ of 0.6 m at 1mm external Na⁺. Decrease of external Na⁺ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na⁺/H⁺ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na⁺/H⁺ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na⁺ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na⁺. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na⁺ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na⁺/H⁺ antiporter to internal protons. The Na⁺/H⁺ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.