Erik Dybing

Inhibitory effects of chlorpromazine and diethylaminoethyl diphenylvalerate (SKF 525-A) on alanine incorporation into protein and α-aminoisobutyric acid uptake in rat hepatoma cells in culture

Abstract Chlorpromazine (CPZ) and SKF 525-A above 0.01 mM inhibit the incorporation of alanine into protein in cultures of a clonal strain of rat hepatoma cells. Fifty per cent inhibition was noted for both drugs at approximately 0.07 mM, and at CPZ 0.15 mM 97 per cent and at SKF 525-A 0.20 mM 96 per cent inhibition respectively was found. Brief washing partially restores normal incorporation rates. CPZ 0.15 mM reduces the amount of total intracellularly accumulated alanine to 11 per cent of controls. The non-metabolizable amino acid α-aminoisobutyric acid (AIB) is actively transported by these cells; CPZ and SKF 525-A inhibit the uptake of AIB, with 44 per cent reduction at a concentration of CPZ 0.15 mM and 55 per cent reduction at SKF 525-A 0.20 mM. The rate of AIB efflux from the cells was also reduced by CPZ 0.15 mM and SKF 525-A 0.20 mM. It is concluded that CPZ and SKF 525-A in a similar pattern inhibit the transport of amino acids in these hepatoma cells in culture.

Glucuronidation in Cultures of Human Skin Epithelial Cells

Abstract. Glucuronidation of p‐aminophenol, p‐nitrophenol and bilirubin has been investigated in cultures of human skin epithelial cells (HE cells) and human skin fibroblasts (FB) and in homogenates from the same cells. HE cells in culture glucoronidated p‐aminophenol and p‐nitrophenol at rates of about 5 and 10 nmol/mg cell protein per h respectively. Bilirubin was not conjugated by HE cells. In homogenates from HE cells supplemented with UDP glucuronic acid (UDPGA) both p‐aminophenol and p‐nitrophenol were glucuronidated at about the same rate as by living cells in culture. Assuming that the transferase activity is the same in the whole cells compared to the homogenates, transferase activity and not UDPGA production may be the limiting factor in glucuronidation of p‐aminophenol and p‐nitrophenol in these cells. The glucuronidation rate of HE cells was not increased by benzpyrene or benzanthrazene. FB did not glu‐curonidate p‐aminophenol, p‐nitrophenol or bilirubin.

Inhibition of acetaminophen glucuronidation by oxazepam.

Abstract Glucuronidation of [ 3 H]acetaminophen (pHAA) in mouse liver microsomes is enhanced about 3-fold by 0.025% Triton X-100. Oxazepam inhibits microsomal glucuronidation of pHAA, yielding apparent competitive kinetics in native microsomes. Phenobarbital-pretreatment has no effect on the microsomal glucuronidation of pHAA. MH 1 C 1 rat hepatoma cells also glucuronidate pHAA, approximately 40 nmoles per mg cell protein per hr being conjugated at a concentration ol 1 mM. Oxazepam also inhibits pHAA glucuronidation in the cell culture system. Intrapcritoneal injection of mice with oxazepam 15 min before subcutaneous injection of pHAA significantly increases the plasma hall-life of pHAA.

Effect of actinomycin D on uptake and incorporation of thymidine and hypoxanthine into the acid-soluble and acid-insoluble fractions of rat hepatoma cells grown in culture

Abstract Preincubation of MH 1 C 1 , rat hepatoma cell cultures with actinomycin D between 0.01 μm/ml and 10.0 μm/ml gives a dose-dependent increase in the uptake of thymidine into the acid-soluble fraction up to 400 per cent of controls; the same increase is found in the acid-insoluble fraction. The effect is detectable after 5 min incubation, but is only fully developed after 1–2 hr treatment with the drug. The stimulation could not be blocked by cycloheximide. Preincubation with actinomycin D has little effect on uridine uptake compared to that of thymidine; actinomycin D 1.0 μm/ml after 2 hr increases uridine uptake to 131 per cent of controls. In contrast the uptake of hypoxanthine is inhibited by actinomycin D, 50 per cent inhibition is seen at 0.75 μm/ml. The apparent K m for the thymidine uptake is 5.9 × 10 −6 M; actinomycin D pretreatment altered the V max of the reaction but did not change the apparent K m . The apparent K m for the hypoxanthine uptake is 5.0 × 10 −6 M; actinomycin D pretreatment gave an apparently noncompetitive inhibition. Actinomycin D does not change the activity of thymidine kinase in homogenates of the cells.

Cycloheximide inhibitation on hypoxanthine transport in cultured cells

Abstract Cycloheximide preincubation inhibits hypoxanthine uptake into the acid-soluble fractions of cultured rat hepatoma cells (MH1C1) and human skin epithelial cells (NCTC 2544, HE cells) in a time- and dose-dependent manner 50% inhibition is seen after 4 h preincubation with 10−4 M cycloheximide of MH1C1 cells and after 2.5 h of HE cells. Adenine uptake is much less affected, after 10 h preincubation with 10−4 M cycloheximide it was reduced to 83% and 67% of controls in MH1C1 cells and HE cells respectively. Cycloheximide inhibits hypoxanthine uptake in a dose-dependent manner above 10−7 M, with 50% inhibition in MH1C1 cells at 4 · 10−7 M after 12 h preincubation and at 10-6 M in HE cells after 6 h preincubation. Puromycin mimics the action of cycloheximide. The inhibition of hypoxanthine uptke is not caused by reduction of the activity of hypoxanthine phosphoribosyltransferase in the two cell lines. 10−4 M cycloheximide preincubation for 10 h does not significantly reduce the uptake of the two non-metabolizable amino acids α-aminoisobutyric acid or 1-aminocyclopentane-1-carboxylic acid (cycloleucine). It is suggested that cycloheximide inhibits the synthesis of a rapidly turning over the protein involved in hypoxanthine transport.

Effects of chlorpromazine and actinomycin D on uptake and incorporation of certain amino acids, hypoxanthine and thymidine in cultures of human skin epithelial cells.

Abstract Chlorpromazine (CPZ) 1· × 10 −4 M inhibited the uptake and incorporation of alanine (25 and 3 per cent of controls respectively), the uptake of α-aminoisobutyric acid (AIB, 39 per cent of controls) and the uptake and incorporation of hypoxanthine (36 and 44 per cent of controls) into acid-soluble and insoluble fractions of human skin epithelial cells (HE cells, NCTC 2544) grown in culture. The uptake of phenylalanine and 1-aminocyclopentane-1-carboxylic acid (cycloleucine) was not inhibited by CPZ in the same dose range, but CPZ above 10 −5 M inhibited the incorporation of phenylalanine into acid-insoluble material with 50 per cent inhibition at 6·5 × 10 −5 M. Actinomycin D stimulated the uptake of thymidine into the acid-soluble fraction of the HE-cells, 5·0 μg/ml increased the uptake to 160 per cent of the controls. The uptake of hypoxanthine was inhibited by actinomycin D, 5·0 μg/ml reduced the uptake to 67 per cent of controls. Actinomycin D did not alter the uptake of AIB or cycloleucine.

Actinomycin D and purine transport in cultured rat hepatoma cells

Abstract Actinomycin D 10−6 M inhibits the uptake of hypoxanthine into the acid-soluble fraction of MH1C1 rat hepatoma cells grown in culture to an average of 20 per cent of controls. Cycloheximide and actinomycin D have additive inhibitory effects on hypoxanthine uptake, whereas the uptake of adenine remains unaffected by actinomycin D. Actinocin, the chromophore of actinomycin D, does not inhibit hypoxanthine uptake. The uptake of guanine by the cells is very low compared with that of hypoxanthine or adenine. Actinomycin D does not change the activity of hypoxanthine-guanine phosphoribosyltransferase in homogenates of the cells. Competition experiments with homogenates of the cells support the view that hypoxanthine and guanine are substrates for the same phosphoribosyltransferase. Whereas adenine is converted to its corresponding nucleoside phosphate by a separate enzyme. Excess adenine inhibits the cellular uptake of hypoxanthine, whereas excess hypoxanthine does not alter the uptake of adenine.

Inhibition of hypoxanthine transport by chlorpromazine and skf 525-A in cell culture

Abstract Chlorpromazine (CPZ, 0.15mM) inhibited the uptake and incorporation of hypoxanthine into TCA-soluble and TCA-insoluble fractions in the MH 1 C 1 rat hepatoma cell line to 38 and 49 per cent of control values, respectively. Diethylaminoethyl diphenylvalerate (SKF 525-A, 0.20 mM) also reduced hypoxanthine uptake and incorporation in the TCA-soluble and TCA-insoluble fractions to 38 and 41 percent, respectively. When nucleic acid synthesis was blocked with actinomycin D and cytosine arabinoside, hypoxanthine uptake was also diminished; CPZ (0.15 mM) further reduced hypoxanthine uptake in the TCA-soluble fraction. The cells showed a time dependent uptake and incorporation of thymidine and uridine; CPZ also inhibited the uptake and incorporation of these nucleosides, but to a much smaller extent than hypoxanthine. CPZ (0.15mM) reduced the thymidine uptake into the TCA-soluble fraction to 71 per cent of controls and those of uridine to 83 per cent of controls. The inhibitory effects of CPZ and SKF 525-A on hypoxanthine and nucleoside uptake and incorporation were equally evident in a fibroblast-derived cell line. It is concluded that CPZ and SKF 525-A inhibit the transport of hypoxanthine and to a lesser degree the transport of thymidine and uridine in the cell cultures.