Uptake and metabolism of polyprenols by animal cells cultured in vitro

Several mammalian, chicken, and mosquito cells grown in vitro take up tritiated dolichol supplied to the incubation medium. The extent of labelling varied markedly between different cell cultures. After 20 h incubation most of the dolichol taken up was unchanged and the major product of metabolism of dolichol was identified as its fatty acid esters. Green-monkey kidney cells were tested with 8 fully unsaturated and 6 alpha-saturated polyprenols ranging from C35 to C105. In general the uptake of alpha-saturated polyprenols (dolichol type, was higher. Considerable differences were found between the uptake of polyprenols of differing chain lengths. Less than 1% of the polyprenols taken up was converted into more polar product, mainly polyprenyl phosphates and polyprenyl phosphate sugars. The short-chain polyprenols, from C35 to C65, were metabolized more rapidly than the long-chain polyprenols, as judged from the amount of polar products and fatty acid esters of polyprenols.     

Uptake and metabolism of glutamine in cultured kidney cells

The metabolism of glutamine was investigated in cultured rat kidney cells. Glutamine utilization and product formation were followed as a function of time at either 10 microM or 1 mM initial glutamine concentration. At 1 mM glutamine, glutamate and gamma-glutamylglutamate were the major products formed at the end of a 5-min incubation period; glutamate accounted for 46% while gamma-glutamylglutamate accounted for 33% of the glutamine utilized. With time, glutamate continued to accumulate while gamma-glutamyl peptide formation leveled off. The role of gamma-glutamyl transpeptidase was assessed by using hippurate, a physiological activator of gamma-glutamyl transpeptidase and acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, an inhibitor of gamma-glutamyl transpeptidase. Hippurate, 4 mM, increased the utilization of glutamine and the formation of glutamate, gamma-glutamyl peptides and ammonia. Exposure of cells to acivicin resulted in 98% inhibition of gamma-glutamyl transpeptidase without effecting phosphate-dependent glutaminase activity. Acivicin inhibition resulted in a decreased utilization of glutamine and product formation as compared to control; 5-oxoproline appearance fell 70%. The fractional distribution of glutamine carbon and nitrogen into its metabolic products in control, hippurate and acivicin-treated cells showed no change at the end of 60 min. The data provide evidence that gamma-glutamyl transpeptidase utilizes glutamine and forms gamma-glutamyl peptides in cultured kidney cells.     

Uptake of creatine by cultured cells

We have examined the uptake of creatine by cultured monolayers of human IMR-90 flbroblasts, human uterine smooth muscle cells, calf aortic smooth muscle cells, and myoblasts and myotubes of the L₆E₉ rat skeletal muscle cell line. Creatine uptake is dependent on temperature and sensitive to the presence of Na⁺ in the extracellular medium. It is saturable, apparently concentrative, and inhibited by ouabain and structural analogs of creatine. In these respects, it resembles the process of creatine uptake by isolated preparations of skeletal muscle and brain tissues. Lineweaver-Burk plots of the data for variation in rate of uptake with concentration of creatine in the medium are nonlinear, suggesting that the process of uptake may be heterogeneous. Assuming the operation of two saturable processes of uptake, we calculated two values for apparent K_m and V for each cell line. Kinetic parameters of creatine uptake by the different cell types are similar. The lower values of K_m (0.02–0.04 mm) are in the physiological range of creatine concentration in mammalian plasma.     

Uptake of hyaluronate by cultured cells

[¹⁴C]hyaluronate is internalized by adsorptive pinocytosis by cultured rat hepatocytes and human synovial cells, but not by human skin fibroblasts and smooth muscle cells. Hyaluronate oligosaccharides compete for the uptake of hyaluronate by hepatocytes without being internalized themselves at the doses used. It is suggested that for adsorptive pinocytosis a hyaluronate molecule has to bind to at least two receptors on the cell membrane.     

Incorporation of exogenous glycosphingolipids in plasma membranes of cultured hamster cells and concurrent change of growth behavior

Globoside added to culture medium was taken up by NIL cells and accumulated as a component of plasma membrane. This was evidenced by the recovery of ³H-labelled globoside from plasma membrane fractions and by the higher chemical quantity of globoside found in NIL cells cultured in medium containing globoside. Concomitantly the following changes in growth behavior were manifested: a reduction in growth rate due to an extended prereplicative phase and a reduced saturation density which may result from changed adhesive properties of cells.     

Uptake of exogenous DNA by pea seedlings and tobacco cells

1.

(1) The uptake of Pseudomonas aeruginosa DNA by pea seedlings, and uptake of tobacco DNA or P. aeruginosa DNA by tobacco cells in shake cultures has been investigated. The fate of the DNA has been followed by CsCl density gradient equilibrium centrifugation, using radiolabeled donor DNA of high density.     

Uptake and release of tetracycline by cultured carrot cells

Summary The nature of tetracycline uptake by carrot cell suspension cultures is described. Tetracycline enters the cells by diffusion and the intracellular level of the antibiotic increases with the amount added. Exposure of carrot cells to high levels of tetracycline for a limited time (24 hr) followed by the removal of the drug and the resuspension of the cells in drug-free medium does not affect cell growth and has no inhibitory effect on protein synthesis (¹⁴C-leucine incorporation).     

Uptake of protein by cultured human trophoblast cells

The uptake of peroxidase by cultured human trophoblast cells was monitored ultrastructurally. There was no apparent effect on the distribution of peroxidase caused by treatment at 4 degrees C, with KCN or with IgG. Pinocytic channels penetrate deep into the perinuclear cytoplasm and may act to allow pinocytosis to occur within layers of microfilaments which might otherwise prevent pinosome access to the majority of the synthetic organelles.     

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarconoma cells Effects of potassium cyanide and dinitrophenol

Cultured monkey hepatocarcinoma cells (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic transformation of the hormones to deiodinated products and their sulfates. When hepatocarconoma cell monolayers were studied at hormone concentrations below 10^?10 M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3′-diiodothyronine and 3′-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol.Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 μM triiodothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3′-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.     

Uptake of transferrin and iron by cultured rat placental cells

This paper describes a method for the culture of rat placental cells. The method involved separation of the basal layer from the labyrinth and sequential digestion of the cells. The cells were demonstrated not to be fibroblasts and are described in terms of their appearance under the light and electron microscopes. Transferrin and iron uptake by the cells was examined and compared with results achieved using other methods of study. The results showed that transferrin bound to receptors on the cell surface and that the transferrin, once bound, was taken into the cell. Only this internalized transferrin was capable of donating iron to the cells. The iron was accumulated within the cells and did not appear to be released to the incubation medium. The apparent dissociation constant (Ka) for transferrin was found to be 6.96 X 10(6) M-1, a value similar to that described by earlier workers. The placental cells had 3.4 X 10(11) binding sites/microgram DNA, equivalent to approximately 1 X 10(6) sites/cell. From these data, and from the rate of accumulation of iron by the cells, the receptor turnover time was estimated as being between 5 and 10 min.     

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells. Effects of potassium cyanide and dinitrophenol

Cultured monkey hepatocarcinoma cell (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic trnasformation of the hormones to deiodinated products and their sulfates. When hepatocarcinoma cell monolayers were studied at hormone concentrations below 10(-10) M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3'-diiodothyronine and 3'-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol. Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 micrometer triidothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3'-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.     

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.     

Metabolism of exogenous polyisoprenoids by animal cells cultured in vitro

The content and type of dolichols in chicken embryo fibroblasts was estimated and the presence of enzymic activities of various dolichyl phosphate sugar transferases was documented. Chicken embryo fibroblasts effectively take up various polyprenoids from the culture medium and catalyse both formation of polyprenyl fatty acid esters and their hydrolysis. With fully unsatured polyprenols the hydrogenation of the terminal alpha-isoprene residue was observed. In baby hamster kidney cells the formation of mannolipids was enhanced by exogenous polyprenols.     

Uptake and accumulation of the herbicide bentazon by cultured plant cells

Cellular absorption of the herbicide bentazon, a weak acid with pK_a 3.45, was investigated using suspension-cultured cells of velvetleaf (Abutilon theophrasti Medic.). Bentazon accumulated rapidly to concentrations approximately four times that of the external medium. Bentazon accumulation against a concentration gradient was not due to its conversion to metabolites, partitioning into lipids, or binding onto cellular constituents. Bentazon uptake was related linearly to the external bentazon concentration, implying that movement of the herbicide into cells was not carrier-mediated. Bentazon was able to diffuse freely and extensively out of the cells, indicating that bentazon can readily diffuse across cell membranes. Potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone inhibited bentazon accumulation as did nitrogen gas when bubbled through the uptake medium. Absorption was pH-dependent with the greatest amount of bentazon accumulating at acidic external pH. Calculations indicated that conversion of uncharged bentazon to bentazon anion in the cytoplasm accounts for cellular accumulation of bentazon. These results provide evidence that bentazon is absorbed across membranes via simple diffusion and that bentazon accumulates in plant cells via an energy-dependent, ion-trapping mechanism which results in bentazon accumulation in the cytoplasm.