Role of glucagon on the control of hepatic protein synthesis and degradation in the rat in vivo.

The effect of glucagon on hepatic protein systhesis and proteolysis has been investigated. The intraperitoneal administration of 200 mug of glucagon produced an increase of the polypeptide chains completion time which was maximal 5 min after its administration and approached control values at 20 min. The increase of the polypeptides chains completion time observed at 5 min after the hormone administration represents a 38% inhibition of the hepatic protein synthetic rate. When glucagon was continuously supplied by intravascular infusion, maximal inhibition was attained throughout the experiment. This inhibition of protein synthesis brought about by glucagon was accompanied by an increase in the polyribosomal state of aggregation, indicating that the hormone acts mainly if not exclusively, on the elongation or termination step, or both. The administration of glucagon produced also a progressive increase in the hepatic valine concentration. This increase could not be accounted for the the decrease in plasma valine levels, suggesting that the rise in haptic valine concentration is an expression of hepatic proteolysis rather than the result of an accelerated transport of amino acids across the hepatocyte plasma membrane. The different time sequence in the glucagon-induced effects of protein synthesis and proteolysis suggests that both effects are independent and probably mediated by different mechanisms.     

Glucagon effect on rat liver protein synthesis in vivo

The in vivo effect of glucagon administration on hepatic polyribosomal profiles has been studied. Glucagon did not change significantly total, free or bound polyribosomal fractions 30–45 minutes after its administration. The combined administration of glucagon plus antiinsulin serum failed to show any significant effect of glucagon over the antiinsulin serum treated control. Glucagon increased valine production in the perfused isolated liver. These results suggest that the well known amino acid catabolic action of glucagon may be preferentially mediated through an increased proteolysis. Since it is known that glucagon increases considerably in vivo the liver cyclic AMP levels then its lack of effect on polyribosomal profiles might indicate that the postulated role for the cyclic nucleotide on liver protein synthesis must be taken cautiously.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.     

Effects of Oxygen Depletion on Norepinephrine- and Carbachol-Stimulated Phosphoinositide Turnover in Rat Brain Slices

We examined the effects of in vitro anoxia and in vivo hypoxia (8% O2/92% N2) on norepinephrine (NE)- and carbachol-stimulated phosphoinositide (PI) turnover in rat brain slices. The formation of 3H-labeled polyPI in cortical slices was impaired by in vitro anoxia and fully restored by reoxygenation. Accumulation of 3H-labeled myo-inositol phosphates (3H-IPs) stimulated by 10(-5) M NE was significantly reduced by anoxia (control at 60 min, 1,217 +/- 86 cpm/mg of protein; anoxia for 60 min, 651 +/- 82 cpm/mg; mean +/- SEM; n = 5; p less than 0.01), and reoxygenation following anoxia resulted in overshooting of the accumulation (control at 120 min, 1,302 +/- 70 cpm/mg; anoxia for 50 min plus oxygenation for 70 min, 1,790 +/- 126 cpm/mg; n = 5; p less than 0.01). The underlying mechanisms for the two phenomena--the decrease caused by anoxia and the overshooting caused by reoxygenation following anoxia--seemed to be completely different because of the following observations. (a) Although the suppression of NE-stimulated accumulation at low O2 tensions was also observed in Ca2+-free medium, the overshooting in response to reoxygenation was not. (b) Carbachol-stimulated accumulation was significantly reduced by anoxia and was restored by reoxygenation only to control levels. Thus, the postanoxic overshooting in accumulation of 3H-IPs seems to be a specific response to NE. (c) The decrease observed at low O2 tensions was due to a decrease in Emax value, whereas the postanoxic overshooting was due to a decrease in EC50 value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of proteolysis during reloading of the unweighted soleus muscle

There is little information on the mechanisms responsible for muscle recovery following a catabolic condition. To address this point, we reloaded unweighted animals and investigated protein turnover during recovery from this highly catabolic state and the role of proteolysis in the reorganization of the soleus muscle. During early recovery (18 h of reloading) both muscle protein synthesis and breakdown were elevated (+65%, P<0.001 and +22%, P<0.05, respectively). However, only the activation of non-lysosomal and Ca(2+)-independent proteolysis was responsible for increased protein breakdown. Accordingly, mRNA levels for ubiquitin and 20S proteasome subunits C8 and C9 were markedly elevated (from +89 to +325%, P<0.03) and actively transcribed as shown by the analysis of polyribosomal profiles. In contrast, both cathepsin D and 14-kDa-ubiquitin conjugating enzyme E2 mRNA levels decreased, suggesting that the expression of such genes is an early marker of reversed muscle wasting. Following 7 days of reloading, protein synthesis was still elevated and there was no detectable change in protein breakdown rates. Accordingly, mRNA levels for all the proteolytic components tested were back to control values even though an accumulation of high molecular weight ubiquitin conjugates was still detectable. This suggests that soleus muscle remodeling was still going on. Taken together, our observations suggest that enhanced protein synthesis and breakdown are both necessary to recover from muscle atrophy and result in catch-up growth. The observed non-coordinate regulation of proteolytic systems is presumably required to target specific classes of substrates (atrophy-specific protein isoforms, damaged proteins) for replacement and/or elimination.     

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.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.     

Phenobarbitone-induced stimulation of protein synthesis in liver of diabetic rat.

The effect of phenobarbitone on liver weight, on the rate of protein synthesis and on the sedimentation profiles of polyribosomes from livers was studied in diabetic rats. The rate of protein synthesis by isolated postmitochondrial supernatants from diabetic rats is lower than that from normal animals. The analysis of polyribosome profiles and the effect of Sephadex chromatography on protein synthesis demonstrated that the reduction was dependent in part on polyribosomal disaggregation and in part on the presence in the cytosol of low molecular weight inhibitor(s). Phenobarbitone administration had the same effect in either diabetic or normal rats in that it increased, (a) the degree of polyribosomal aggregation, (b) the rate of protein synthesis by the isolated postmitochondrial supernatants, (c) liver weight and (d) the activity of the inducible enzyme, NADPH-cytochrome c reductase. Both polyribosomal and soluble factors appear to be involved in the phenobarbitone effect. As the diabetic rats do not secret insulin the results suggest that insulin is not involved in the control of protein synthesis by phenobarbitone. It is suggested that the intracellular redox state has a major influence on the rate of protein synthesis.     

Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.     

Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited.     

Metabolism of hypoxanthine in isolated rat hepatocytes.

The hepatic metabolism of hypoxanthine was investigated by studying both the fate of labelled hypoxanthine, added at micromolar concentrations to isolated rat hepatocyte suspensions, and the kinetic properties of purified hypoxanthine/guanine phosphoribosyltransferase from rat liver. More than 80% of hypoxanthine was oxidized towards allantoin; less than 5% of the label was incorporated into the purine mononucleotides, and a similar proportion appeared transiently in inosine. The maximal velocity of oxidation (approx. 750nmol/min per g of cells) was in close agreement with the known activity of xanthine oxidase in liver extracts. In contrast, the maximal velocity of the incorporation of labelled hypoxanthine into mononucleotides reached only 30nmol/min per g of cells, compared with an activity of hypoxanthine/guanine phosphoribosyltransferase, measured at substrate concentrations analogous to those prevailing intracellularly, of 500nmol/min per g of cells. Hypoxanthine incorporation into the mononucleotides was decreased by allopurinol, anoxia and ethanol, despite inhibition of its oxidation under these conditions; it was increased by incubation of the cells in supraphysiological concentrations of Pi. Allopurinol and anoxia decreased the concentration of phosphoribosyl pyrophosphate inside the cells by respectively 40 and 60%, ethanol had no effect on the concentration of this metabolite and Pi increased its concentration up to 10-fold. The kinetic study of purified hypoxanthine/guanine phosphoribosyltransferase showed that a mixture of ATP, IMP, GMP and GTP, at the concentrations prevailing in the liver cell, decreased the V max. of the enzyme 6-fold, increased its Km for hypoxanthine from 1 to 4 microM and its Km for phosphoribosyl pyrophosphate from 2.5 to 25 microM. In the presence of 5 microM-hypoxanthine and 2.5 microM-phosphoribosyl pyrophosphate, the mixture of nucleotides inhibited the activity of purified hypoxanthine/guanine phosphoribosyltransferase by 95%. It is concluded that this inhibition results in a limited participation of hypoxanthine/guanine phosphoribosyltransferase in the control of the production of allantoin by the liver.     

The effects in vitro of hypoglycaemia and recovery from anoxia on synaptosomal metabolism.

Synaptosomes from several regions of the rat brain were found to exhibit half-maximal rates of 14CO2 output and [14C]acetylcholine synthesis from D-[U-14C]glucose at glucose concentrations approx. 50-fold lower than those required by the brain in situ. However, synaptosomal acetylcholine synthesis was found not to be directly proportional to substrate oxidation as measured by 14CO2 output. When synaptosomes had been exposed to anoxia in vitro, their metabolic indices (14CO2 and [14C]acetylcholine synthesis, and adenine nucleotide levels) were found not to be significantly different from control aerobic values, unless they had been subjected to veratridine depolarization. This is in accord with previous findings that neither the absolute metabolic rates nor the vulnerability to hypoxic damage exhibited by brain in situ is reflected by brain slices in vitro, unless these are stimulated by depolarization. The use of synaptosomes as a model for synaptic damage in vivo is discussed.     

FURTHER EVALUATION OF POLYPEPTIDE SYNTHESIS IN CEREBRAL ANOXIA, HYPOXIA AND ISCHEMIA

The alteration of polypeptide synthesis was evaluated with microsomes isolated from anoxic rabbit, hypoxic rat and ischemic gerbil brains to estimate the extent of functional or structural changes in polyribosomes in situ and the extent of artifact during tissue preparation. By using two-stage experimentation with combination of control and pathological microsomes and supernatant, it was found that the previously observed effects on microsomal or polyribosomal polypeptide synthesis in the above pathophysiological conditions were mainly the reflection of the alteration of polyribosomes in situ rather than the artifact during tissue preparation by degradative processes. In support of this finding. the use of inhibitors of degradative enzymes did not significantly protect microsomes either in normal or in pathological conditions. It was noted that the decline of tissue pH, to a certain extent, could be correlated with dysfunction of polyribosomes both in situ and during tissue preparation in cerebral hypoxia and anoxia. Since there is little change in ATP level, it was postulated that the alteration of pH in situ is responsible for the observed suppression of polypeptide synthesis in vitro at least in cerebral hypoxia. This hypothesis was supported by the subsequent experiments with incubation of brain slices and homogenization of brain tissue under various pH. It was emphasized that the environmental biochemical elements surrounding polyribosomes in cytoplasm should be evaluated as possible contributing factors for polyribosomal dysfunction in such pathological conditions as cerebral anoxia, hypoxia or ischemia if the alteration of energy state does not explain the phenomenon entirely.     

Glycogen Metabolism in Neonatal Rat Brain During Anoxia and Recovery

Abstract: Metabolic alterations in glycogen and in glycogen-related metabo lites were studied in neonatal rat brain during controlled anoxia and recovery. One-day postnatal rats were exposed to 100% N, at 37°C for up to 20 min; some rats were allowed to recover in air. Animals were frozen in liquid N, and the brains were prepared for fluorometric analysis of compounds involved in glycogen turnover. During anoxia, glycogen decreased by 29% and 42% at 10 and 20 min, respectively; the free (soluble) and bound (insoluble) components of glycogen decreased in nearly equal proportions. Brain glucose decreased by 72% at 10 min with little further change there after; G-6-P, G-1-P, and UDPG also declined. During recovery from anoxia, glucose and G-6-P increased above control levels for up to 60 min. G-1-P paralleled G-6-P levels, but UDPG remained low. Glycogen returned to control values by 4 h. The findings suggest that although glycogen is mobilized slowly in newborn rat brain, the metabolite contributes at least one-third of the cerebral energy supply during anoxia. Presumably, readily available stores of glycogen combined with low cerebral metabolic requirements underscore the known tolerence of immature animals to hypoxic stress. Glycogen accumulation during recovery appears to be facilitated at the synthetase step, since equilibrium measurements of the phosphoglucomutase and pyrophosphorylase systems indicate that these reactions are not rate-limiting for glycogen synthesis.     

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.     

Inotropic effects of adrenaline on the anoxic or hypercapnic myocardium of rainbow trout and eel

Summary The effects of adrenaline on the development of force under anoxia and hypercapnic acidosis (13% CO₂ in 30 mM HCO ₃ ^– ) were examined in isolated, electrically stimulated cardiac ventricle strips of rainbow trout and eel.During anoxia or acidosis applied 15 min in advance, the adrenaline concentration of the bathing solution was increased in 5 steps from 0 to 10^–4 M with 5 min at each step. Before levelling off the contractile tension increased by 145±42% (mean±SE) in the anoxic, 80±14% in the acidotic and 152±41% in the control trout cardiac strips. Except for the acidotic strips the corresponding values tended to be lower for the eel strips being 46±9%, 57±17% and 57±9%, respectively. The inotropic affinity for adrenaline was lower in the trout than in the eel myocardium. For the trout myocardium it remained unchanged, while it decreased somewhat for the eel myocardium under anoxia or acidosis.Adding to the muscle bath 10^–5 M adrenaline resulted in an increase in force development by about 90% for the trout myocardium and 50% for the eel myocardium. 5 min later anoxia or hypercapnic acidosis was applied for 30 min followed by 30 min at control conditions. Relative to the force values recorded just before anoxia or acidosis was applied, the changes in contractile force during these periods were the same with and without adrenaline. Thus adrenaline appears to have a persistent positive inotropic effect in the fish myocardium during and after oxygen lack or acidosis.     

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.