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 microM VBL and 500 microM VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 microM 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 microM for VBL and 110 microM for VCR. Since the Ki 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.

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 degrees 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 7.0 +/- 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 degrees 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.

Induction of HL-60 cell differentiation by 3-deaza-(+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase.

The metabolically stable inhibitor of S-adenosylhomocysteine hydrolase (AdoHcyase), 3-deaza-(+/-)-aristeromycin (dzAri) has recently been shown to induce differentiation in HL-60 cells. The present study was undertaken to characterize the cytostatic, cytotoxic, and differentiation inducing properties of dzAri in HL-60 cells and to investigate biochemical consequences of AdoHcyase inhibition. A dye exclusion test and a clonogenic assay were used to test cytotoxic and cytostatic properties. dzAri had reversible cytostatic effects on HL-60 cells at concentrations lower than 10 microM and partially reversible cytotoxic effects above 10 microM. The induction of differentiation was dependent upon concentration and time of exposure, with maximal effect after 6 days incubation with 5-10 microM dzAri. Washout experiments demonstrated that the cells were not committed to differentiation after 48 h of incubation with dzAri. The AdoHcyase of HL-60 cells was inhibited with a Ki of 20 nM. The concentration of S-adenosylhomocysteine increased dose dependently 48 h after incubation with 0.1-100 microM dzAri. The incorporation of [3H]methyl from [methyl-3H]methionine into 5-methylcytosine of DNA was reduced by 26% at 5 microM dzAri. The findings indicate that continuous presence of dzAri is necessary to induce differentiation and inhibit proliferation in HL-60 cells. The inhibition of AdoHcyase perturbs levels of transmethylation metabolites and the incorporation of [3H]methyl into 5-methyl-cytosine of DNA.

Effects of anaesthetics on protein synthesis in isolated rat hepatocytes: inhibition by diethyl ether in contrast to no influence by pentobarbital and fentanyl.

Hepatocytes were isolated from livers of fasted rats by a two-step Ca++-free/collagenase perfusion method. Suspensions of parenchymal liver cells were incubated in the absence and presence of three different anaesthetics, diethyl ether, pentobarbital and fentanyl at various concentrations. Their influence on the hepatocytes was monitored by measuring protein synthesis as the incorporation of L-(U-14C) valine (50 mCi/mol, 4.2 mM) into liver proteins. Diethyl ether representing anaesthetics mainly affecting cellular membranes unspecifically, inhibited protein synthesis markedly, concentrations of approximately 10, 20 and 30 mM caused 27, 50 and 74 per cent inhibition respectively, of cellular protein synthesis. The rate of synthesis process of these proteins or that ether also inhibited protein secretion from cells to media. The effect of diethyl ether was completely reversible when the anaesthetic was removed by changing the medium. Pentobarbital representing barbiturate anaesthetics, reduced the synthesis of cell and medium proteins very little, while the opiate anaesthetic fentanyl had no inhibitory effect. These results demonstrate a potential hepatotoxic mechanism for membrane active drugs like diethyl ether. They also indicate that special precautions should be taken when this type of anaesthesia is used during the study of hepatic protein synthesis.

Reduced synthesis of hepatic and plasma proteins in rats during diethyl ether anaesthesia.

The effect of diethyl ether anaesthesia on in vivo hepatic protein synthetic rates was tested in male Wistar rats. Protein synthesis was measured by an isotope technique after correction for measured levels of precursor specific radioactivity. It was shown that usual anaesthetic levels of diethyl ether reduced the rate of synthesis of liver proteins with 20% compared to a group receiving no anaesthesia. The synthesis/secretion of plasma proteins was much more inhibited, with approximately 70-80%, compared to animals either receiving no anaesthesia or pentobarbital. Monitoring of ether concentrations therefore seems necessary in experiments in which the hepatic capacity for protein synthesis/secretion is measured.

No effect of acute ethanol administration on hepatic protein synthesis and export in the rat in vivo.

Ethanol was administered as a single p.o. dose (2.88 g X kg-1) to male rats (220-265 g body weight) to give blood alcohol concentrations of 40-50 mM for the following 3 hr. Controls were given isoenergetic amounts of either sucrose or lipid. Liver protein synthetic rates were measured during a 20 min interval at the end of the 3 hr period following the administration of diets. Although ethanol caused a 32% reduction of the incorporation of labelled valine into liver protein compared to the sucrose group during the 20 min interval, no such reduction was found when the synthetic rate of stationary liver protein was calculated (182 vs. 214 (not significant) pmol X mg protein-1 X min-1) for same interval. There was no difference between the ethanol and lipid group with regard to either incorporation or synthetic rates. Incorporation of valine into plasma proteins was reduced in the ethanol group compared to the sucrose group, but not compared to the lipid control group, again demonstrating no ethanol-specific effect. When the incorporation into plasma proteins was divided by the specific radioactivity of valyl-tRNA at 20 min, the difference between the ethanol and the sucrose group disappeared. The fraction of newly synthesized proteins exported to the plasma measured 40 min after the injection of labeled valine, was equal in all three treatment groups. It was concluded that acute administration of ethanol has no consistent effect on liver protein synthesis and secretion in vivo.

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.

Acute effects of halothane and enflurane on drug metabolism and protein synthesis in isolated rat hepatocytes.

The metabolism of sulphanilamide, antipyrine and paracetamol was studied in the absence and presence of the anaesthetics halothane and enflurane at three different concentrations (0.5, 1.0 and 2.0 mM) in isolated hepatocytes from the rat. Cell viability and protein synthesis were monitored to evaluate toxic effects. A strong concentration related inhibition of antipyrine oxidation (40-70%) and paracetamol conjugation (20-40%) was caused by both halothane and enflurane. Acetylation of sulphanilamide was not inhibited, however, as a slight augmentation was noticed. A significant dose related decrease of cell viability (3-13%) was caused by both anaesthetics. Dose dependent inhibition of the synthesis of stationary cell proteins (15-60%) and the synthesis/secretion of medium proteins (35-85%) was caused by halothane. Similar but slightly less pronounced effects were caused by enflurane. The present findings show that volatile anaesthetics may have general effects as well as different degrees of specific effects on both membrane bound enzyme and soluble enzyme activities.

A study of hepatic protein synthesis, three subcellular enzymes, and liver morphology in chronically ethanol fed rats.

Male Wistar rats were given ethanol chronically (20-30% of the energy as ethanol) in a nutritionally sufficient regimen. Controls received lipid as isoenergetic substitute for ethanol. Treatment lasted for 2 or 8 weeks. Hepatic protein synthesis was measured in fasted rats during a 32 min. continuous infusion of 3H-valine. After 2 weeks of treatment accumulation of hepatic protein was observed in the ethanol group, but there was no change in hepatic protein synthesis or morphology. After 8 weeks the rate of hepatic protein synthesis was decreased by 35% in the ethanol group, but there was no accumulation of protein and a slight accumulation of intracellular lipid droplets. Neither the subcellular distribution of incorporated 3H-valine, nor the activities and distributions of alcohol dehydrogenase and NADPH cytochrome c reductase were changed. Mitochrondrial cytochrome c oxidase activity was decreased in the ethanol group, and cytosolic and microsomal fractions showed higher cytochrome c oxidase activity in this group. Chronic ethanol treatment for 8 weeks had an adverse effect on general protein synthesis as well as on a specific enzyme in the liver in the absence of serious morphologic abnormalities.

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.

Characterization of S-adenosylhomocysteine binding to isolated rat hepatocytes and purified rat liver plasma membranes. Effect of analogues of S-adenosylhomocysteine.

Studies on the disposition of extracellular S-adenosylhomocysteine by isolated rat hepatocytes have shown that S-adenosyl-L-homocysteine is not taken up by cells, but binds to acceptor(s) on the cell surface. The Scatchard plots for the binding of S-adenosylhomocysteine to hepatocytes and purified rat liver membranes at 0 degrees were nonlinear, and consistent with high-affinity components with Kd values of 0.4 microM and 0.7 microM, respectively. About 60% of the S-adenosylhomocysteine that was bound to cells and purified membranes dissociated rapidly from its binding sites. The rapid initial phase was followed by a second slow phase obeying first-order kinetics, corresponding to a dissociation rate constant of 0.09 min-1. S-Tubercidinylhomocysteine and unlabeled S-adenosylhomocysteine were potent inhibitors of the binding of S-[14C]adenosylhomocysteine, whereas S-3-deazaadenosylhomocysteine, S-adenosylmethionine, and S-adenosyl-D-homocysteine were less effective. A fraction of the S-adenosylhomocysteine that was bound to rat hepatocytes was displaced by low concentrations of sinefungin and its metabolite, A9145C, but these compounds were weak inhibitors of S-adenosylhomocysteine binding to purified membranes. 5'-Deoxy-5'-S-isobutylthioadenosine showed slight inhibitory activity against S-adenosylhomocysteine binding to both cells and purified membranes. In conclusion, the equilibrium binding, dissociation rate kinetics, and displacement curves in the presence of S-adenosylhomocysteine analogues show that S-adenosylhomocysteine binds to a heterogeneous population of binding sites of intact hepatocytes and purified liver plasma 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.

Inhibition of fracture healing by indomethacin in rats.

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 microgram/ml in the indomethacin-treated animals. In the long-term experiment, indomethacin inhibited fracture healing (P less than 0.006) and increased the angulation between the femur fragments. In the short-term experiments indomethacin inhibited fracture healding (P less than 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).

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.

Metabolism of 14C-antipyrine in suspensions of isolated rat liver cells.

Suspensions of liver cells isolated from perfused rat livers were incubated with antipyrine-N-methyl-14C. Antipyrine was eliminated by first-order kinetics during incubations for 3 hours with primary suspensions (parenchymal cells + non-parenchymal cells) and suspensions of purified parenchymal cells. Antipyrine concentrations were unchanged when incubated with suspensions of non-parenchymal cells, dead cells or medium only. At the end of incubation period, 4-OH-antipyrine and 3-CH2OH-antipyrine were detected mainly as the glucuronide or sulphate conjugates, and evidence for the N-demethylation of antipyrine was also obtained. Half-lives for elimination of antipyrine in primary cell suspensions were not significantly different from the half-lives measured in parenchymal cell suspensions. This finding together with the lack of metabolism of antipyrine found in non-parenchymal cell suspensions suggest that oxidation and conjugation of antipyrine is mainly confined to the parenchymal cells. There was significant inhibition of antipyrine metabolism in primary suspensions by phenylbutazone (1.6 X 10(-3)M), dexamethasone (2 X 10(-4)M) and ethanol (1.3 X 10(-2)M, 0.75%). We suggest that the use of primary suspensions of isolated rat liver cells provide a rapid and simple method for the study of factors influencing drug metabolism in the liver.

Progesterone-binding globulin and testosterone-binding activity in guinea pig serum during pregnancy: relationship to progesterone and oestrogens.

Progesterone levels in serum have been determined throughout pregnancy in guinea-pigs by a competitive protein-binding technique, using pregnant guinea pig plasma protein as binding agent. The concentrations of this protein (progesterone-binding globulin, PBG) as well as the testosterone-binding activity (TBA) have been quantitated by means of equilibrium dialysis. In order to correlate these parameters to the endogenous oestrogen production, the urinary oestrogen excretion was recorded. The concentration of progesterone, PBG and TBA showed a sharp rise on days 14-18 of gestation, reaching a maximal level on days 30-44. The progesterone level thereafter declined significantly towards parturition, followed by a sharp drop post partum. PBG and TBA followed a similar course, the decline towards parturition, however, being non significant. Neither progesterone, PBG nor TBA were significantly correlated to the urinary oestrogen excretion. The concentration of PGB was remarkably high, amounting to 1-2 x 10(-5) M during most of the guinea pig pregnancy. This binding capacity generally exceeded the endogenous progesterone concentration by a factor of 20, as calculated on a molar basis. A mean of 64% of the binding sites was available for testosterone binding, corresponding to a free binding capacity of 400 mug testosterone per 100 ml.