Furosemide-sensitive potassium efflux in cultured mouse fibroblasts

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.     

Multinucleation of cultured canine epithelial and lymphoma cell lines

The frequency distribution patterns of monuclear and multinuclear giant cells were determined for two canine lymphoma cell lines (DT-5 and 11028), and a normal canine kidney epithelial cell line (DK). The proportion of multinuclear cells in the DK line (1.53%) was approximately twice those of the DT-5 (0.75%) and 11028 (0.73%) cell lines. The observed frequency distributions of cells with single and various numbers of multiple nuclei were compared to Poisson distributions using the chi-square test. For each cell line, the number of cells with three or more nuclei far exceeded the number predicted by the Poisson distribution. Hence, the occurrence of multinuclear cells in these canine cell lines does not follow a random distribution pattern. Possible explanations for the nonrandom accumulation of multinuclear giant cells are discussed.     

Expression of human growth hormone in cultured mouse fibroblasts

The new system for the transfer and expression of foreign genes based on retroviral vectors pPS-neo, conferring neomycin resistance was constructed. The BALB/c mouse cell lines producing highly active human growth hormone (more than 7 micrograms/ml into culture medium) were constructed using these vectors. An antibody column was used to purify the growth hormone from cell culture medium. Possibilities of producers to be applied for gene therapy are discussed.     

Cation transport in murine L fibroblasts cultured long term in a serum-free medium

Cation fluxes and intracellular content in mouse fibroblasts L, growing for more than three years in the Dulbecco modified Eagle's serum-free medium (clone L625sf) were measured as a function of culture density. The cells show no density-dependent inhibition of growth and in continuously growing cultures of L625sf cells internal potassium, and rubidium influx was found to remain high within a wide range of densities (5.10(4)-20.10(4) cells/cm2). A close correlation was revealed between the potassium transport and the proliferative state of L625sf cultures: the addition of 5% calf serum to logarithmically growing cultures leads to the increase in culture growth rate as well as to the increase in ouabain-inhibited rubidium influx and intracellular potassium content; a delay in culture growth rate due to medium depletion is accompanied by decreasing both the rubidium influx and the intracellular potassium content. It is concluded that L625sf cells being capable of multiplicating in serum-free medium remain sensitive to growth factors of serum and may be used for study of growth factor induction of cell proliferation and for identification of autocrine factors of cell growth.     

Stimulation by insulin of DNA synthesis in cultured mouse fibroblasts]

With the aid of autoradiography, the effect of insulin on entering S- from G1-period of the mitotic cycle and on the rate of DNA synthesis of the mouse fibroblasts (L), was studied,--in the cells incubated for 24 hr in serum-free medium. In these conditions the cells were temporarily blocked in G1-period. Insulin (100 mcU/ml) increased by 1.5-fold the amount of cells in S-period as well as caused a marked stimulation of DNA synthesis.     

Hypovitaminosis-A in the mouse prostate gland cultured in chemically defined medium

Prostate glands of young adult mice were grown by the organ culture technique in natural and defined medium, in air and in an atmosphere of 95 per cent O₂ and 5 per cent CO₂.Under all conditions the alveolar structure of the organ was preserved in vitro.In glands kept in natural medium the alveoli were lined with secretory epithelium, the cells of which were higher and better preserved in the centre of the explant in cultures kept in oxygen and CO₂ than in those kept in air.In glands grown in the defined medium in both air and oxygen, irregularly distributed foci of squamous metaplasia appeared in the majority of explants.The metaplasia was either preceded by atrophy, or by hyperplasia and stratification of the secretory lining epithelium, and in many explants both squamous and secretory epithelium were present in the same alveolus.The squamous changes could be completely suppressed by treatment with vitamin A and it is concluded that they are caused by vitamin A deficiency of the tissue cultured in the defined medium which does not contain this vitamin.     

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts

Summary It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrinclot retractile (FCR) activity, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2–4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.     

Effect of antioxidants on cultured human diploid fibroblasts exposed to cystine-free medium

Logarithmically growing human embryonic diploid cells started to die in cystine-free medium within 18 hours. Glutathione accounted for almost all the acid-solube sulfhydryl compound of the cells and cellular glutathione level decreased rapidly after cystine depletion. By adding vitamin E the cells survived over 6 days in cystine-free medium, though glutathione content of the cells was reduced to less than 1% of the normal level. Synthetic antioxidants had similar effect, and mechanism by which cells die in cystine-free medium was suggested.     

Hydralazine stimulates production of oxygen free radicals in Eagle's medium and cultured fibroblasts.

The effect of hydralazine on the oxygen free radical production was studied in whole cultured murine liver fibroblasts and mitochondrial and microsomal fractions of the cells by ESR spin trapping with DMPO and measurement of Tiron semiquinone formation. Hydralazine itself was found to generate free radicals in phosphate buffer and especially in Eagle's Minimal Essential Medium. Most of the adduct of the spin trap DMPO was due to its reaction with hydralazine-induced hydroxyl radical. Moreover, this compound stimulated free radical formation in fibroblasts. These data suggest that hydralazine alters the cellular free radical metabolism which may have implications for the biological activity of this drug.