Potassium fluxes and ouabain binding in growing,density-inhibited and rous sarcoma virus-transformed chicken embryo cells

Potassium fluxes, ouabain binding, and Na⁺ and K⁺ intracellular concentrations were determined for cultures of growing normal, density-inhibited and Rous sarcoma virus-transformed chicken embryo fibroblasts. No significant differences in K⁺ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus-transformed cells; however, ouabain binding and ouabain-sensitive K⁺ influx were 1.5- to 1.8-fold lower in density-inhibited cells. Thus, potassium influx in this system can be classified as a growth-related, but not transformation-specific change. As determined by both flame photometry and radioisotopic (⁴²K) equilibration, growing normal and density-inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4-fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2- to 4.5-fold higher in transformed than normal or density-inhibited cells. Complementary studies of potassium efflux showed a 1.3- to 1.5-fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density-inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

The effect of harmaline on unidirectional potassium fluxes and ouabain binding in renal cell cultures

Harmaline inhibits K⁺ influx into primary cell cultures of ground squirrel kidneys to a greater extent than either ouabain or furosemide. A concentration of 200 μM harmaline was required to inhibit half of the total K⁺ influx; this effect was also seen at low temperature (5°C), and in another species (hamster). Although kinetic analysis of K⁺ influx indicates that harmaline does not compete with extracellular K⁺, harmaline did reduce the binding of [³H]ouabain to the cells. K⁺ efflux was also reduced. Therefore, harmaline may inhibit the furosemide-sensitive Na⁺/K⁺ cotransport system as well as the ouabain-sensitive Na⁺/K⁺ pump.     

The effects of sodium and potassium on ouabain binding by human erythrocytes

Ouabain binding by the human erythrocyte membrane is reversible, exhibits a high degree of chemical specificity, and can be detected at ouabain concentrations as low as 1 x 10^-10 M. The relation between ouabain binding and ouabain concentration can be described by a rectangular hyperbola permitting determination of the maximal binding (B _max) and the ouabain concentration at which ouabain binding is half-maximal (K_B). Reducing the external sodium concentration increased K_B, while reducing the external potassium concentration decreased K_B. Neither cation altered B _max The reciprocal of K_B was a linear function of the sodium concentration at sodium concentrations ranging from 0 to 150 mM. Conversely, the relation between the reciprocal of K_B and the external potassium concentration was nonlinear, and raising the potassium concentration above 4 mM produced no further increase in K_B. These results are compatible with a model which postulates that the erythrocyte membrane contains a finite number of receptors each composed of a glycoside-binding site and a cation-binding site. When sodium occupies the cation-binding site, the affinity of the glycoside site for ouabain is increased; when potassium occupies the cation-binding site the affinity of the glycoside site for ouabain is decreased.     

Secreted proteins of quiescent,serum-stimulated and over-confluent mouse embryo fibroblasts

Quiescent and proliferating cultures of Swiss mouse embryo fibroblasts were pulse labelled with [¹⁴C]-amino acids and the newly synthesized proteins that were secreted into the medium were resolved by electrophoresis on Polyacrylafde gradient gels. Conditioned media obtained from quiescent cultures that were stimulated to grow by the addition of 20% fetal calf serum showed the presence of two unique polypeptides of molecular weights 48000 and 26000. A polypeptide of molecular weight 45000 was present in increased amounts in serum-stimulated cells than in quiescent cells. This protein was also superinduced in quiescent cells by cycloheximide treatment. Mouse embryo fibroblasts grown under over-crowded conditions secreted two proteins of molecular weights 35000 and 11000. The 35 K polypeptide was shown to be related to the major excreted protein of transformed cells, since it was immunoprecipitated by an antiserum to major excreted protein. These results indicate that the 48 K and 26 K proteins may be proliferation specific proteins, while the 35 K protein present in the conditioned media of over-confluent cells may be a marker of morphological transformation.     

Ubiquitin in stressed chicken embryo fibroblasts

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.     

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.     

Serum stimulation of sodium transport in human fibroblasts containing low and high levels of intracellular sodium

Summary The relationships between intracellular sodium content, sodium transport and serum effects were investigated in human fibroblasts. In the cells with low intracellular sodium (Na _iL ^/+ ;0.04 mol sodium/mg protein) serum stimulated the sodium-potassium pump as measured by ouabain-sensitive sodium efflux and rubidium influx and also exerted a transstimulation of ouabain-insensitive sodium transport resulting in net influx. In cells with high intracellular sodium (Na _iH ^/+ ;0.42 mol sodium/mg protein) all aspects of sodium transport were increased compared to Na _iL ^/+ cells. In these cells serum caused no change in sodium-potassium pump activity but significantly increased the ouabain-insensitive sodium fluxes resulting in net efflux. In Na _iL ^/+ cells, serum promoted net sodium influx through an amiloride-sensitive pathway that was undetectable in the basal state. In Na _iH ^/+ cells the serum-stimulated net efflux was amiloride sensitive but this pathway also contributed to a major portion of sodium transport in the basal state. This study demonstrated that sodium-potassium pump activity is directed by the supply of internal sodium and that serum can increase this supply by promoting net influx, and that serum-induced sodium transport can be modified by intracellular sodium content.     

DNA repair and survival in UV-irradiated chicken embryo fibroblasts

The role of the pyrimidine dimer in cell killing, DNA synthesis and repair has been studied by utilizing the light-requiring DNA-repair mechanism of photo- reactivation in UV-irradiated chicken-embryo fibroblasts. Survival, as measured by colony-forming ability at 41°C, is increased in cells left in the light. The initial inhibition of DNA synthesis by UV is much less in light-treated cells, and levels reach that of unirradiated controls much faster than when the cells are left in the dark. The number of endonuclease-sensitive sites (dimers)_measured by an assay with a crude extract from M. luteus, rapidly decreases as the cells are allowed to photoreactive. However, in the dark, significant amounts of repair also occur, but at a much lower rate and with a lag phase of several hours. Unscheduled DNA synthesis occurs to a similarly low extent in both dark- and light-treated cells, confirming the finding that some amount of excision repair occurs that is light-independent. When survival is examined as a function of the number of dimers present, the dimers, not the non-dimer products, appear to be responsible for cell killing. In this study, the removal of dimers in vivo by photoreactivation has made it possible to demonstrate directly that dimers are primarily responsible for the deleterious effects of UV on DNA synthesis and survival.     

Furosemide-sensitive sodium and potassium fluxes in human neonatal erythrocytes

During the course of incubation, in vitro, in a saline medium, we found an increase of cell volume and Na+ content in human neonatal erythrocytes (NNE), from the umbilical cord. The increased cell volume was dependent on the major anion in the medium in that replacement of Cl- by NO-3 abolished the cell volume increase. In erythrocytes from adults neither the cell volume nor the sodium content were altered under similar incubation conditions. Furosemide-sensitive Na+ and K+ fluxes were at variance from those reported from adult erythrocytes. The differences here presented between both cell types would be another instance of changes observed to occur in erythrocytes during the postnatal period.     

Isolation of clonal strains of chicken embryo fibroblasts

A method for cloning of chicken embryo fibroblasts (CEF) was developed, yielding a cloning efficiency of up to 50% without use of feeder cells or conditioned medium. An analysis of the growth potential of over 200 randomly selected clones showed that only approx. 4% of the clones were capable of doubling more than 35 times before undergoing cellular senescence. A positive correlation between initial growth rate and in vitro lifespan was observed. This served as a basis for a simple selection procedure for fibroblast strains suitable for long-term culturing. None of over 200 clones thus isolated could be established into a line. Subclones from clonal CEF strains were more homogeneous than uncloned CEF cultures with respect to morphology and growth behaviour, but still heterogeneous in their in vitro life span. All fibroblast strains tested could be effectively infected and transformed by a variety of avian sarcoma and leukosis viruses.     

Kinetics and thermodynamics of ouabain binding by intact turkey erythrocytes: effects of external sodium ion, potassium ion, and temperature

The kinetics of association and dissociation for the ouabain-Na+,K+- dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na+ concentration, K+ concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-first-order kinetics with an association rate constant (k1) that is conveniently determined over a wide range of temperatures (5-37 degrees C). The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-1) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5 degrees C. Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na+ concentration and falling with increasing K+ concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant k1, k-1 in contrast being unaffected at any given temperature by even very large changes in Na+ or K+ concentration. Arrhenius plots of k1 and k-1 both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: delta G degree = -9.8 kcal/mol; delta H degree = +5.3 kcal/mol; delta S degree = +48.7 cal/degree/mol. The large positive value of delta S degree presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of delta H degree.     

鸡胚成纤维细胞cDNA表达文库的构建

鸡胚成纤维细胞(CEF)是研究鸡传染性法氏囊病病毒(IBDV)的主要细胞材料,而构建CEF的cDNA表达文库是筛选IBDV在CEF中的细胞受体,研究细胞嗜性的基础平台。采用Gateway技术构建CEF的表达文库,避免使用限制性内切酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。该技术将CEF的mRNA分离纯化后,以5′端生物素标记的Oligo(dT)primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×106cfu/mL,文库总容量为1.2×107cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×105cfu/mL,文库总容量为5.5×106cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为研究病毒受体和病毒入侵途径,进一步了解IBDV的致病机理奠定了基础。     

Decreased ouabain binding in cystic fibrosis fibroblasts in potassium-free medium

We have previously demonstrated that cystic fibrosis (CF) cells show increased survival compared to normal cells when exposed to ouabain in medium lacking potassium. In this report, we show that the CF cells bind significantly less ouabain than the normal cells in potassium-deficient medium. Using age-matched normal and CF skin fibroblast strains, we show that (1) at ouabain concentrations <20 × 10⁻⁹ M binding for both normal and CF cells is linear with time for 1 h and reaches equilibrium after 4 h; (2) at ouabain concentrations between 2 and 20 × 10^-9 M, the initial rate of binding for CF cells is approx. 70% of the normal cells; (3) under equilibrium-binding conditions, Scatchard analysis reveals that three different CF strains have 12, 16, and 44% fewer ouabain-binding sites than their matched normal controls. In addition, studies in potassium-free medium of the inhibition of ⁸⁶Rb flux (a K⁺ analogue) into the cell by ouabain show no differences between CF and normal cells. We have also previously shown that in a low glucose, potassium-deficient medium the CF cells survived ouabain exposure no better than normal cells. In this report, equilibrium-binding studies with [³H]ouabain clearly show that CF cells bind less ouabain under these conditions than normal cells. These results indicate that ouabain resistance in CF cells is not solely a function of differences in ouabain binding. Furthermore, the differential ouabain killing may not be due to ion transport differences, but rather to as yet unknown mechanisms. CF cells thus appear to be unlike previously characterized ouabain-resistant mutants.     

Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum-arrested and serum-stimulated cells. Addition of platelet-derived growth factor to serum-arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000-dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5-6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.