Inhibition of protein synthesis by (aminooxy)acetate in rat liver

(Aminooxy)acetate and D-cycloserine, two inhibitors of hepatic transamination reactions, inhibited also protein synthesis in isolated cells and postmitochondrial supernatants from rat liver. Both inhibitors acted in extracts only in concentrations higher than 1 mM. However, while D-cycloserine acted in isolated cells, as in extracts, (aminooxy)acetate inhibits protein synthesis in isolated cells by 50% of the control in the range 0.03-3 mM. NH+4 and H2O2, two by-products of (aminooxy)acetate degradation, inhibited protein synthesis in isolated liver cells, but at such a high concentration that the inhibition of protein synthesis carried out by (aminooxy)acetate cannot be explained by generation of these species. The results point out that the inhibitory action of (aminooxy)acetate on protein synthesis appears to require the integrity of the molecule.     

Failure of a branched chain amino acid-enriched diet to reverse ethanol inhibition of cardiac protein synthesis in the rat

The fractional rate of protein synthesis was determined in the hearts of rats in vivo fed on diets containing 27% of energy as ethanol or on this diet supplemented with 5% of equimolar amounts of branched chain amino acids (BCAA). Administration of ethanol significantly decreased the fractional synthetic rate of mixed cardiac proteins and this depression was not ameliorated by concomitant feeding of BCAA. These data are discussed in relation to the stimulation of cardiac protein synthesis by BCAA observed in vitro.     

Inhibition of protein synthesis by Cl-

Optimum K+ concentration for protein synthesis in four eukaryotic cell-free systems is obtained with 70 to 80 mM added KCl or with 110 to 150 mM added K(OAc). The different K+ optima are due to inhibition of protein synthesis by Cl- at concentrations higher than those present in the cytoplasm of eukaryotic cells. Initiation of protein synthesis is severely inhibited with 150 mM added KCl. This inhibition results from an impairment of mRNA binding to ribosomes. The binding of initiator Met-tRNAt, however, is only slightly inhibited by 150 mM KCl.     

Inhibition of protein synthesis by glucagon in different rat muscles and protein fractions in vivo and in the perfused rat hemicorpus.

The effect of glucagon on the rate of muscle protein synthesis was examined in vivo and in the isolated perfused rat hemicorpus. An inhibition of protein synthesis in skeletal muscles from overnight-fasted rats at various plasma concentrations of glucagon was demonstrated in vivo. The plantaris muscle (Type II, fibre-rich) was more sensitive than the soleus (Type I, fibre-rich). Myofibrillar and sarcoplasmic proteins were equally sensitive in vivo. However, protein synthesis in mixed protein and in sarcoplasmic and myofibrillar fractions of the heart was unresponsive to glucagon in vivo. In isolated perfused muscle preparations from fed animals, the addition of glucagon also decreased the synthesis of mixed muscle proteins in gastrocnemius (Type I and II fibres) and plantaris, but not in the soleus. The sarcoplasmic and myofibrillar fractions of the plantaris were also equally affected in vitro. Similar results were observed in vitro with 1-day-starved rats, but the changes were less marked.     

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachement completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Inhibition of protein synthesis on homologous and heterologous desensitization in the rat adipocyte

Desensitization of lipolysis was induced in isolated rat adipocytes by incubation with isoproterenol 10^?5M or ACTH 250 mU/ml for two and three hours, respectively. Those cells desensitized with isoproterenol were restimulated with either isoproterenol 10^?7M or ACTH 6 mU/ml and those cells desensitized with ACTH were restimulated with isoproterenol 10^?7M. Lipolysis was quantitated by the release of cyclic AMP and glycerol. No effect on either homologous or heterologous desensitization was observed when either cycloheximide 2 μg/ml or puromycin 10^?4M was included in the incubation media during the induction of desensitization. These findings support the conclusion that protein synthesis plays no role in the desensitization of lipolysis in the isolated rat adipocyte.     

Acute effects of ethanol in the control of protein synthesis in isolated rat liver cells

The acute effect of ethanol on hepatic protein synthesis is a rather controversial issue. In view of the conflicting reports on this subject, the effect of ethanol on protein labeling from l-[³H]valine in isolated liver cells was studied under a variety of experimental conditions. When tracer doses of the isotope were utilized, ethanol consistently decreased the rate of protein labeling, regardless of the metabolic conditions of the cells. This inhibition was not prevented by doses of 4-methylpyrazole large enough to abolish all the characteristic metabolic effects of ethanol, and it was not related to perturbations on the rates of l-valine transport and/or proteolysis. When ethanol was tested in the presence of saturating doses of l-[³H]valine no effect on protein labeling was observed. These observations suggest that the ethanol effect in decreasing protein labelling from tracer doses of the radioactive precursor does not reflect variations in the rate of protein synthesis but reflects changes in the specific activity of the precursor. These changes probably are secondary to variations in the dimensions of the amino acid pool utilized for protein synthesis. Even though it showed a lack of effect when tested alone, in the presence of saturating doses of the radioactive precursor ethanol inhibited the stimulatory effects on protein synthesis mediated by glucose and several gluconeogenic substrates. This effect of ethanol was not prevented by inhibitors of alcohol dehydrogenase, indicating that a shift of the NAD system to a more reduced state is not the mediator of its action. It is suggested that ethanol probably acted by changing the steady-state levels of some common effector(s) generated from the metabolism of all these fuels or else by preventing the inactivation of a translational repressor.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors.

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Inhibition of lipid synthesis in Bacillus amyloliquefaciens by inhibitors of protein synthesis

Inhibitors of protein synthesis reduce the level of lipid production in Bacillus amyloliquefaciens, both in growing cells and in non-dividing washed-cell suspensions. This effect is primarily on phospholipids rather than acetone-soluble lipids. Although the cells contain five major phospholipids the washed-cell supernatants contain only one phospholipid species whose accumulation is sensitive to chloramphenicol but insensitive to rifampicin.     

Inhibition of N-methyl-D-aspartic acid-nitric oxide synthase in rat hippocampal slices by ethanol

The ionotropic glutamatergic receptor system, especially the subtype mediated by N-methyl-D-aspartic acid (NMDA), is known to exhibit special sensitivity to the effect of ethanol. This is due partly to the ability of ethanol to modulate the production of nitric oxide through the NMDA-nitric oxide synthase (NOS) pathway. In this study, we examined the effects of ethanol on basal and NMDA-stimulated NOS activity in rat hippocampal slices by measuring the conversion of [(14)C]-arginine into [(14)C]-citrulline in an incubation system containing the necessary cofactors. Stimulation of hippocampal slices with NMDA (100 microM) enhanced NOS activity by 43% (n = 12). Although ethanol did not alter NOS activity when added to the incubation system during NMDA stimulation, it dose-dependently inhibited NMDA-NOS activity when added to the slices during the 90-min preincubation period. Further assay of NOS activity with brain cytosolic fraction indicated an inhibitory effect of ethanol (200 mM) when the assay was carried out in the absence of exogenous tetrahydrobiopterin (BH4), a redox-active cofactor for NOS. Incubation of brain homogenates resulted in a time-dependent increase in the levels of lipid peroxidation products, but ethanol did not further enhance these products. Taken together, these results provide evidence for the role of BH4 but not oxidative stress in the inhibitory effect of ethanol on NMDA-NOS activity in rat hippocampal slices.