Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180 000 sites/cell but this fell to 125 000 (p <0.001) in cells enriched in monounsaturated fatty acids and rose to 386 000 (p <0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants ( and ) of 0.03 and 0.01 nM⁻¹. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM⁻¹ whereas values of 0.01 and 0.005 nM⁻¹ were obtained in the cells enriched in polyunsaturated fatty acids (all comparison p <0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.     

Effect of magnesium-dependent cell membrane alterations on the transport of K+ in Ehrlich ascites tumour cells

Mg-deficiency or Mg-loading of tumour cells changes the permeability of the cell membrane. The influence of this change on the K+ transport across the membrane was investigated using 86Rb+ and K+ analog. The time course of the influx and efflux rates were estimated by means of a mathematical approach for a two-compartment system with inconstant pool sizes. The comparison of the two states of the cells demonstrates that in Mg-deficient cells the passive K+ efflux is significantly enhanced (40%). This in turn stimulates the active counter transport mediated by the (Na+-K+)-ATPase, raising the ATP consumption by about 30%. However, the enzyme is not able to maintain the cellular K+ content under these conditions. After a short transient increase due to the initially enhanced influx the passive net efflux prevails. Differences in the electrophoretic mobility of the two states of the cells confirm Mg-dependent changes of the cell membrane structure.     

Lanthanum-induced alterations in cellular electrolytes and membrane potential in Ehrlich ascites tumor cells

We have investigated the effect of varying La⁺³ concentrations (0.01 mM to 2.0 mM) on membrane potential and electrolyte composition of Ehrlich ascites tumor cells. La⁺³ concentrations less than 0.02 mM had no effect. Above 0.02 mM, La⁺³ induced concentration-dependent loss of electrolytes and water from the cells. At 1.0 mM the effect was maximal and resulted in an 87% reduction in cellular K⁺, 79% in Cl^? and 21% in Na⁺ within 4.8 minutes. The Na⁺ loss occurred even in the face of an electrochemical potential gradient favoring Na⁺ entry. La⁺³ increased the recorded values of membrane potential; the magnitude of the effect was related to the external La⁺³ concentration, and was maximal at 1.0 mM. Studies using ¹⁴⁰La showed that La⁺³ binds rapidly to the cell surface and does not enter the cells. The amount of La⁺³ bound to the cells was related to the external La⁺³ concentration by a sigmoidal curve and was maximal at about 1.0 mM. The bound La⁺³ could not be displaced by either added La⁺³ or Ca⁺². Agents known to effect the integrity of the cell membrane, such as phospholipase C, neuraminidase, pronase and Hg⁺² were tested for their ability to displace bound La⁺³. Only pronase displaced bound La⁺³, indicating that La⁺³ associates with cell protein. It is hypothesized that La⁺³ rapidly interacts with membrane protein causing alterations in membrane permeability and capacity to actively transport ions.     

Effect of the diabetic environment on the lipid metabolism of Ehrlich ascites cells

One strain of Ehrlich ascites cells lacking of insulin receptors, was grown into control and diabetic mice and cells harvested from diabetic mice reimplanted into control mice. The fatty acid composition of neutral and polar lipids was analyzed and several parameters calculated. Results showed that it is possible to produce similar changes in the lipid fatty acid unsaturation of Ehrlich cells to those observed in the liver of the diabetic bearing mice. These changes may be reverted by growing these cells into control mice. The diabetic environment also promoted a relative increase in the radioactivity from incorporated in vitro into neutral lipids of Ehrlich cells. This metabolic adjustment, probably due to an induction of the enzyme diglyceride acyltransferase, was completely reverted by transplanting these cells in control mice. The metabolic adaptation of Ehrlich ascites cells to the diabetic environment did not modify their biological behaviour as pointed out by their mean generation time. The evidence presented here, showing relatively normal growth of Ehrlich cells in association with changes in the lipid fatty acid pattern and in lipid metabolism, indicates the adaptation of these cells, lacking of insulin receptors, to the environment provided by the diabetic mice.     

Effects of membrane lipid unsaturation on the interactions of insulin and multiplication stimulating activity with endothelial cells

Modification of plasma membrane fatty acyl composition has resulted in major changes in insulin binding and insulin action in several cell types. In the present study, endothelial cells, which in vivo are directly bathed by the changing fatty acid and insulin environment of the bloodstream, were grown in media enriched in specific saturated, monounsaturated and polyunsaturated fatty acids. These media conditions resulted in major and specific alteration in fatty acyl unsaturation of both neutral lipids and phospholipids of the endothelial cells. Despite the extensive fatty acyl changes, the lipid-modified cells demonstrated no change in the binding of insulin or the insulin-like growth factor, multiplication stimulating activity, and little alteration in insulin-induced down-regulation of the insulin receptor, or in cell processing of insulin. We suggest that the insulin receptor of the endothelial cell responds in a different manner than other cell types to similar alterations of membrane fatty acyl composition.     

Concanavalin A-induced alterations in sodium and potassium content of Ehrlich ascites tumor cells

Net fluxes of sodium and potassium were studied in Ehrlich mouse ascites tumor cells during contact with the agglutinating protein, concanavalin A. This lectin altered cation transport markedly at concentrations of 20–105 μg/ml (6–47 μg/mg cell protein). Whereas control cells extruded sodium and maintained or accumulated potassium against electrochemical gradients, in the presence of concanavalin A there was rapid net sodium entry and potassium loss. After 10–20 minutes in concanavalin A, sodium extrusion began and potassium loss diminished but these events were prevented by ouabain. The alterations in cation content induced by concanavalin A are unlikely to be the result only of agglutination since soybean agglutinin caused much smaller changes although it agglutinated the cells equally well.     

Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Lanthanum-induced alterations of cellular electrolytes in Ehrlich ascites tumor cells: a new view

We have shown previously that Ehrlich ascites tumor cells maintained at room temperature under an oxygen atmosphere lose Na⁺, K⁺ and Cl^? isosmotically when exposed to La⁺⁺⁺ (0.1 to 1.0 mM). Concomitant with these changes there is an increase in the recorded membrane potential (increasing intracellular negativity). The present studies further characterize the effect of La⁺⁺⁺ on electrolyte distribution. Ehrlich ascites tumor cells were maintained at 0.5° C to permit Na⁺ gain and K⁺ loss. The addition of 1 mM La⁺⁺⁺ to low temperature cells induces rapid loss of Na⁺, K⁺ and Cl^?. This net loss of cellular electrolytes occurs even in cells depleted of ATP content using 2-deoxyglucose (5 mM) and rotenone (10^?6 M ). Analysis of the appearance of tracer ²²Na in the environment of cells preloaded with the radioisotope shows that La⁺⁺⁺-induced changes in membrane permeability or in active ion transport mechanisms are not responsible for the dramatic loss of electrolytes from experimental cells. The electrolyte loss occurs only when the cells are resuspended mechanically during the washing procedure used to prepare the cells for electrolyte determination. We conclude that the results of La⁺⁺⁺ interaction with Ehrlich ascites tumor cells are twofold. As we have previously reported, La⁺⁺⁺ stabilizes and causes a hyperpolarization of the membrane potential. Secondly, La⁺⁺⁺ predisposes the cell membrane to become highly permeable when subjected to mechanical stress.     

Mitoxantrone toxicity on Ehrlich ascites tumour cells: inhibition of the transplasma membrane redox activity

This study focused on the potent cytotoxic effect that mitoxantrone produces on Ehrlich ascites tumour cells. Host mice treated with mitoxantrone showed a life span three times higher than control non-treated host mice. Mitoxantrone also showed a potent cytotoxic effect on Ehrlich cells incubated in vitro for only a few hours. Studies on the effect of mitoxantrone on a plasma membrane redox system showed that mitoxantrone inhibits this activity, which is apparently related to cell proliferation.     

Inorganic phosphate in Ehrlich ascites tumor cells and its distribution across the cell membrane

A regulatory function of the cell membrane in controlling the cytoplasmic level of Pi has been proposed, and in Ehrlich ascites tumor cells an active influx of primary phosphate has been reported in the literature. In the present study, Ehrlich cells were incubated at 1.5--50 mM extracellular Pi at pH 7.4 (Pi mainly secondary phosphate) and at pH 6.0 (mainly primary phosphate), and the measured cell Pi was compared with the value expected from a passive distribution of Pi. At a low extracellular Pi concentration the cell Pi was 3--6 mumol/g or even more. It is suggested that a major part of this cell Pi can be accounted for by enzymic release of Pi during the sampling procedure. If this interpretation is correct, the present results show that both ionic species of Pi are in electrochemical equilibrium across the cell membrane at steady state. Moreover, in vivo the concentration of free Pi in the cytosol will presumably be maintained at a steady-state level of about 0.4 mM, one order of magnitude below the directly measured values. This implies that the ratio [ATP]/[ADP][Pi] which is important in the regulation of energy metabolism, is higher than reported in the literature.