Atle Bessesen

Inhibition of fracture healing by indomethacin in rats

Abstract. The effect of indomethacin (2 mg/kg/day) on the healing of closed unimmobilized femoral fractures was examined in rats. A standard femoral fracture was produced in 205 male adolescent rats, and three different experiments were done. In a long‐term experiment, the rats were treated with either indomethacin or placebo for 29 days and fracture healing followed for a maximum of 91 days. In two short‐term experiments, the rats were treated with either indomethacin or placebo for a week and followed for a maximum of 122 days. The effect of age was studied in one experiment. Indomethacin plasma levels were about 1 μg/ml in the indomethacin‐treated animals. In the long‐term experiment, indomethacin inhibited fracture healing (P< 0·006) and increased the angulation between the femur fragments. In the short‐term experiments indo‐methacin inhibited fracture healing (P< 0·033) and increased the interfragmentary angle as well as fracture instability. All untreated fractures healed within 10 weeks in younger rats (210 g), whereas only 44% healed in older rats (295 g).

Inhibition of protein synthesis by ethanol in isolated rat liver parenchymal cells.

Abstract Hepatocytes were isolated from perfused rat livers. 0.8 · 106–5.0 · 106 cells/ml were incubated at 37°C in the absence and presence of ethanol (50 mM). 1. (1.) The incorporation of l -[U-14C]valine and l -[3,5-3H2]tyrosine into both stationary cell protein and proteins released to the medium was reduced by ethanol. 2. (2.) This effect was neither mediated by reduced uptake of labelled valine nor diminished specific radioactivity of the precursors. 3. (3.) The effect was present in amino acid-free media, making possible effects of ethanol on uptake of other amino acids an unlikely explanation of the reduced incorporation. More probably, ethanol primarily affects the protein-synthesizing machinery of the liver cells. 4. (4.) No effects of ethanol were found when liver cell proteins were prelabelled with l -[U-14C]valine and the radioactivity remaining in the protein after inhibition of further protein synthesis with cycloheximide (5 μg/ml) was measured. In other prelabelling experiments performed without cycloheximide, ethanol did not affect the amount of radioactive valine remaining within and released from liver protein. It was concluded that ethanol reduced hepatocyte protein synthesis without affecting protein degradation. Hepatocytes appear more sensitive to this effect of ethanol than intact liver preparations, when the present observations are compared with previous results.

Incorporation of labelled amino acids into proteins of isolated parenchymal and nonparenchymal rat liver cells in the absence and presence of ethanol

Parenchymal and nonparenchymal cells were isolated from perfused rat livers and incubated at 37 degrees C in the absence and presence of ethanol (50 mM). 1. Nonparenchymal cells prepared by means of centrifugation showed a higher rate of incorporation of L-[U-14C]valine into protein than nonparenchymal cells prepared by means of pronase. Cells prepared by the former method were used for further studies. 2. Protein degradation was present in suspensions of both parenchymal and nonparenchymal cells evidenced by increasing levels of branched amino acids in the intracellular and extracellular compartment during cell incubation. 3. The rate of cellular protein synthesis (corrected for precursor pool specific radioactivity) was of the same order of magnitude in nonparenchymal and parenchymal cells when expressed as nmol valine incorporated per mg protein. This rate was also close to the value found in intact liver by other workers. 4. Approximately 25% of the total radioactivity incorporated during incubation for 2 h was found in proteins released to the medium from parenchymal cells, while the corresponding figure for nonparenchymal cells was 3.5%. 5. Ethanol inhibited incorporation of labelled valine into stationary and medium proteins of parenchymal cells. No such effects were found in nonparenchymal cells. 6. Nonparenchymal cells did not metabolize ethanol while parenchymal cells did, shown by changes in lactate/pyruvate ratio and medium pH. It was concluded that nonparenchymal cells are capable of synthesizing proteins at a rate comparable to that found in parenchymal cells. Protein synthesis in parenchymal cells was inhibited by ethanol, but nonparenchymal protein synthesis was unaffected. This difference may be linked to the ability of the former cell type to metabolize ethanol.

Ethanol and protein metabolism in the liver

The influence of acute and chronic ethanol administration on liver protein synthesis, secretion and degradation has been studied by various research groups. Acute ethanol administration appeared to have few if any effects on protein synthesis in vivo, but reduced the synthetic rates of both stationary and exported proteins in suspensions of isolated rat liver cells. Chronic ethanol intake for more than 4 weeks inhibited protein synthesis in vivo, and in cell preparations from treated rats. This inhibitory effect was independent of animal sex, hepatic protein content and diet. The effects of acute and chronic ethanol intake on hepatic protein export are unclear with both inhibition or no effect being reported. The effect of ethanol on liver protein degradation has only been studied to a limited extent, and the results do not indicate clear and marked effects due to ethanol. The inhibitory effect of chronic ethanol intake on hepatic protein synthesis could be of importance in the development of liver injury.

Differentiation of Human Leukemia Cells by Nucleoside Analogues

Induction of differentiation in human leukemia cells by nucleoside analogues was first reported by Lotem and Sachs in the promyelocytic cell line HL-60 (1). At concentrations causing a 50% inhibition of cell growth, bromodeoxyuridine (BrdU) changed cell morphology, and cytosine arabinoside (ara-C) and BrdU changed the number of Fc and C3 rosettes. A wider range of nucleosides was tested in HL-60 cells, by means of morphology and nitroblue tetrazolium test (NBT) by Bodner, Ting and Gallo (2). 3-Deazauridine (c3urd), was the most potent while 5-azacytidine (5-aza-Cyd) (for review see ref. 3) was only moderately effective. Morphological criteria indicated that the nucleosides had effects on neutrophil maturation comparable to effects of retinoic acid at concentrations of about 20 uM.

Distribution of nortriptyline in human blood: effects of temperature, pH, and drug concentration

An in vitro study of putative factors causing postvenipuncture redistribution of the tricyclic antidepressant nortriptyline (NT) in blood was carried out in blood samples from a single subject. The influence of varying temperature, pH, and drug concentration on the protein binding of NT was assessed by equilibrium dialysis. The influence of the same variables on the distribution of NT in whole blood was measured by calculating the cell/plasma (C/P) drug concentration ratios after centrifugation of blood samples. Variation in temperature did influence the distribution of NT in blood considerably. From 4 to 37°C, the C/P ratio was found to increase from 1.0 ± 0.1 to 1.7 ± 0.2, and protein binding expressed as unbound fraction from 2.9 ± 0.2 to 7.1 ± 1.1% (mean ± SD). Neither pH variations within the 7.0–7.6 range, nor variations in drug concentration, were found to alter NT distribution at 37°C. The observed effect of temperature on NT distribution shows that handling of blood samples from patients taking NT should be standardized in order to avoid errors of interpretation.

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 μM VBL and 500 μM VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 μM VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (Ki values) of 55 μM for VBL and 110 μM for VCR. Since theKi values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.

Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: Relation between binding and phospholipid methyltransferase activity

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.

Formation of Acetaldehyde from Diethyl Ether in Man

Diethyl ether anaesthesia caused detectable blood acetaldehyde levels in 15 patients. The average acetaldehyde concentration was 21 micro M which approximates the level found after intake of ethanol. No acetaldehyde could be found in patients anaesthetised without ether.

Binding of S-adenosylhomocysteine to isolated rat hepatocytes and purified plasma membranes from rat liver

Extracellular AdoHcy, the endogenous transmethylase inhibitor formed from AdoMet during transmethylation, is not taken up by rat hepatocytes (Aarbakke & Ueland, 1981). On the other hand, accumulation of intracellular AdoHcy is associated with a pronounced export of AdoHcy, suggesting a vectorial transport of AdoHcy (Hoffman et al., 1980). AdoHcy may thus interact with a permease in the cell membrane. Additional observations suggesting interaction of AdoHcy with membrane components, include: inhibition of membrane transport by the AdoHcy analogue, SIBA (Pierre & Robert-Gero, 1979); binding of AdoHcy to the surface of rat hepatocytes (Aarbakke & Ueland, 1981) and membranes from rat cerebral cortex (Fonlupt et al., 1981); and inhibition by AdoHcy of phospholipid methyltransferase of rat liver plasma membrane (Schanche et al., 1981). The present communication reports on the characteristics of AdoHcy binding to isolated rat hepatocytes and purified rat liver plasma membranes.