Regulation of chondrosarcoma metabolism by cyclic adenosine 3'5'-monophosphate

The in vitro effects of cyclic AMP on amino acid transport and synthesis of macromolecules in the Swarm rat chondrosarcoma were investigated using the cyclic AMP analogue, N6-monobutyryl cyclic AMP (MBcAMP), and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). Amino acid transport was assessed by measuring alpha-amino-[1-14C]isobutyrate (AIB) uptake. The synthesis of macromolecules was estimated by measuring radiolabeled precursor incorporation into total proteins, proteoglycan, and RNA. MBcAMP stimulated [14C]AIB uptake, [3H]uridine transport, and UTP formation. MBcAMP inhibited 35SO4 and [3H]leucine incorporation into proteoglycan and stimulated [3H]uridine incorporation into RNA. MIX elevated endogenous cyclic AMP levels in the Swarm rat chondrosarcoma and mimicked the effects of MBcAMP on AIB transport and radiolabeled precursor incorporation into macromolecules. For comparative purposes, the effects of MBcAMP on AIB uptake and macromolecule synthesis in rat costal cartilage segments were investigated. MBcAMP and MIX stimulated AIB uptake by costal cartilage segments, inhibited [3H]leucine incorporation into total protein and 35SO4 incorporation into proteoglycan, and had no effect on [3H]uridine incorporation into RNA.     

Nitric oxide-dependent protein synthesis in parotid and submandibular glands of anaesthetized rats upon sympathetic stimulation or isoprenaline administration

In anaesthetized female rats, the beta-adrenoceptor agonist isoprenaline was intravenously infused (20 microg kg(-1) min(-1)) for 30 min or the ascending cervical sympathetic nerve trunk was intermittently stimulated (50 Hz, 1 s every tenth second) on one side for 30 min. The incorporation of [3H]leucine into trichloroacetic acid (TCA)-insoluble material was used as an index of protein synthesis. In response to isoprenaline, the [3H]leucine incorporation increased by 79% in the parotid glands and by 82% in the submandibular glands. The neuronal type NO-synthase inhibitor N-PLA, reduced (P < 0.001) this response to 26% and 20%, respectively. Sympathetic stimulation under alpha-adrenoceptor blockade increased the [3H]leucine incorporation by 192% in the parotid glands and by 35% in the submandibular glands. N-PLA reduced the corresponding percentage figures to 86% (P < 0.01) and 8% (P < 0.05). When tested in the parotid glands, the non-selective NO-synthase inhibitor L-NAME reduced (P < 0.01) the nerve-evoked response to 91%. The increase in [3H]leucine incorporation in response to sympathetic stimulation under beta-adrenoceptor blockade was not affected by N-PLA in the parotid (139% versus 144%) and submandibular glands (39% versus 34%). In non-stimulated glands, the [3H]leucine incorporation was not influenced by the NO-synthase inhibitors. In conclusion, beta-adrenoceptor mediated salivary gland protein synthesis is largely dependent on NO generation by neuronal type NO-synthase, most likely of parenchymal origin.     

Role of leucine in protein metabolism during exercise and recovery.

Exercise produces changes in protein and amino acid metabolism. These changes include degradation of the branched-chain amino acids, production of alanine and glutamine, and changes in protein turnover. One of the amino acid most affected by exercise is the branched-chain amino acid leucine. Recently, there has been an increased understanding of the role of leucine in metabolic regulations and remarkable new findings about the role of leucine in intracellular signaling. Leucine appears to exert a synergistic role with insulin as a regulatory factor in the insulin/ phosphatidylinositol-3 kinase (PI3-K) signal cascade. Insulin serves to activate the signal pathway, while leucine is essential to enhance or amplify the signal for protein synthesis at the level of peptide initiation. Studies feeding amino acids or leucine soon after exercise suggest that post-exercise consumption of amino acids stimulates recovery of muscle protein synthesis via translation regulations. This review focuses on the unique roles of leucine in amino acid metabolism in skeletal muscle during and after exercise.     

Insulin regulation of fat cell ribosomes, protein synthesis, and lipoprotein lipase

Ribosomes of high purity were isolated from fat cells by discontinuous sucrose gradient centrifugation. Approximately 23% of the ribosomes were membrane bound. A reproducible fraction of ribosomes was recovered as polysomes capable of incorporating [3H]leucine into peptides in a cell-free system. Insulin (0.1-1.0 mU/ml) produced an increase in polysomal activity. Linear sucrose gradient profiles also revealed an increase in the ratio of polysomes to total ribosomes. Coincident with this effect, insulin increased the lipoprotein lipase activity fraction inhibitable by cycloheximide (0.01 mg/ml), and the immunotitratable enzyme activity. Insulin also enhanced the incorporation of labeled amino acids into adipose tissue immunoprecipitable lipoprotein lipase and total fat cell proteins. Cordycepin (0.1 mg/ml) or alpha-amanitin (10 micrograms/ml) partially inhibited insulin effects on ribosomes and protein synthesis but not on lipoprotein lipase. EGTA (1mM) prevented all of the insulin effects, whereas the calcium ionophore A-23187 (2 microM) augmented the hormone actions. The ionophore alone partially mimicked the insulin effects. In adipocytes, insulin increased the size of the protein-synthesizing polysomal pool by a mechanism which, in part, requires nuclear mRNA processing. Insulin, however, increased lipoprotein lipase synthesis independent of nuclear events. Calcium ions may be important for the expression of these insulin effects.     

Aromatic amino acid incorporation into proteins in rat brain slices: influences of other amino acids affecting transport

Incorporation of [³H]tryptophan, [³H]tyrosine and [³H]phenylalanine into proteins has been studied in cerebral coronal slices from adult and 1-day-old rats. Unlabelled amino acids greatly increased (histidine) or decreased (leucine, phenylalanine, tyrosine, tryptophan) intracellular levels of the [³H]amino acids, but only slightly decreased their incorporation rates. Effects on uptake were similar in experiments with adult and newborn rats, but patterns of protein synthesis inhibition seemed to differ. Amino acid uptake and subsequent incorporation into proteins were thus not closely associated. Incorporation in slices from adult rats was much slower than in those from newborn rats, and moreover, one half of it was cycloheximide-insensitive. The different inhibition patterns by other amino acids may thus be explained by multiple mechanisms in cerebral amino acid incorporation into proteins.     

Effect of lanthanum on pancreatic protein synthesis in streptozotocin-diabetic rats

The role of extracellular Ca2+ in mediating the stimulatory effect of cholecystokinin octapeptide (CCK8) on [3H]phenylalanine incorporation into protein was studied in isolated pancreatic acini from streptozotocin-diabetic rats. The stimulatory effect of CCK8 (10(-10) M) on [3H]phenylalanine incorporation was completely abolished by preincubating acini with either 10(-4) M lanthanum or 10(-3) M manganese. At these concentrations neither compound altered the basal rate of amino acid incorporation, and both compounds inhibited CCK-mediated Ca2+ influx without affecting either basal or CCK-mediated 45Ca2+ efflux. Lanthanum (10(-4) M) also blocked the stimulatory effect of the cholinergic analogue carbachol (10(-5) M) on amino acid incorporation but did not alter the stimulatory effect of insulin (1.67 x 10(-8) M). Vasoactive intestinal polypeptide and 12-O-tetradecanoyl-phorbol-13-acetate failed to increase the incorporation of [3H]phenylalanine into acinar protein. These findings suggest that CCK and other pancreatic secretagogues that act via Ca2+ enhance protein synthesis by increasing cell membrane permeability to Ca2+ and provide additional evidence that this may be an important mechanism by which CCK regulates pancreatic exocrine function.     

Effect of acute exercise on amino acid incorporation into the rat myocardium

Summary To determine the effect of acute exercise and recovery on amino acid incorporation into myocardial protein, female Wistar rats (220–240 g) were exercised to exhaustion by swimming. Animals were sacrificed at exhaustion, or 0.5, 1, 2, 4, or 16 h after exercise. Hearts were removed and perfused for 30 min with a Krebs-Henseleit bicarbonate buffer containing; 15 mM glucose, normal plasma levels of amino acids and 0.1 Ci [³H] phenylalanine per ml of buffer. Immediately following the exhaustive exercise, amino acid incorporation into extramitochondrial, mitochondrial and whole heart protein was decreased by 50, 55, and 43% respectively. Following 2 h of recovery, incorporation of [³H]-phenylalanine returned to normal in all three protein fractions. No stimulation in protein synthesis was observed in any of the cell fractions. In an attempt to estimate the intracellular availability of [³H]-phenylalanine, the TCA soluble radioactivity was determined. No change from rest was observed at exhaustion or throughout recovery suggesting that the amino acid pool size was not altered. These data indicate that exhaustive exercise temporarily reduces myocardial protein synthesis which quickly returns to normal during recovery.     

Incorporation of leucine into human skeletal muscle proteins.

The in vitro incorporation of labelled leucine into human skeletal muscle proteins was studied with the aim to elucidate the relationship between the amino acid tissue pools and protein biosynthesis. The distribution volumes of leucine and cycloleucine in skeletal muscle tissue were similar but the equilibration time was shorter for leucine than for cycloleucine. The cellular uptake of leucine and cycloleucine was competitively inhibited by increased concentration of amino acids in the medium indicating an active transport. Optimal stimulation for incorporation of leucine into proteins was obtained at an amino acid concentration in the medium corresponding to 10 times that of normal human plasma. The incorporation of 14C-leucine into skeletal muscle proteins was linear before the total pool of free intracellular 14C-leucine and the incorporation rate of leucine calculated from the specific activity in the medium versus the amino acid concentration in the medium were different in the same experiment indicating a re-utilization of amino acids released at protein degradation. The results are compatible with the hypothesis that the proteolytically released amino acids have a competitive advantage for incorporation as compared with extra- and intracellular free amino acids. It is concluded that the amino acid pool which is in the immediate continuity with the protein biosynthesis sites equilibrates rapidly with the extracellular amino acid pool.     

Effect of exercise on amino acid incorporation into myocardial contractile proteins

Amino acid incorporation into myocardial protein was studied in rats after an acute bout of exhaustive swimming. Hearts were removed at exhaustion, 1, 2, or 4 h of recovery and amino acid incorporation measured using [³H] phenylalanine in an isolated perfused heart preparation. Amino acid incorporation into total tissue protein was reduced 30% at exhaustion but returned to normal by 1 h of recovery and showed no further change 4 h post exercise. In the myofibrillar fraction amino acid incorporation decreased slightly after exhaustive exercise but was stimulated by 57% following 2 h recovery. Myosin, electrophoretically fractionated showed an 84% stimulation in phenylalanine incorporation at exhaustion and 112% increase 2 h post exercise. Amino acid incorporation into myosin light chains (LC₁ and LC₂) accounted for most of the increased rate of synthesis. These data suggest that there was a preferential increase in myocardial protein synthesis following exercise which was associated with the myosin light chain components of the contractile proteins.     

Inositol stimulates DNA and protein synthesis, and expansion by rabbit blastocysts in vitro.

The effect of different concentrations (0, 0.6, 3, 15, 75 and 375 microM) of myo-inositol on the development of rabbit morulae to expanded blastocysts was investigated in terms of blastocyst expansion and synthesis of DNA and protein, as measured by incorporation of [3H]thymidine and [14C]amino acids into acid-precipitable material. A concentration of 15 microM inositol caused a 2.8-fold increase in blastocyst expansion (P less than 0.01), a 9.9-fold increase in thymidine incorporation into DNA (P less than 0.01) and a 3.6-fold increase in amino acid incorporation into protein (P less than 0.01). There were no significant differences in the range from 15 to 375 microM inositol.     

Acute effects of acidosis on protein and amino acid metabolism in perfused rat liver

Acidosis is frequently associated with protein wasting and derangements in amino acid metabolism. As its effect on protein metabolism is significantly modulated by other abnormal metabolic conditions caused by specific illnesses, it is difficult to separate out the effects on protein metabolism solely due to acidosis. The aim of the present study was to evaluate, using a model of isolated perfused rat liver, the direct response of hepatic tissue to acidosis. We have compared hepatic response to perfusion with a solution of pH 7.2 and 7.4 (controls). Parameters of protein and amino acid metabolism were measured using both recirculation and single-pass technique with 4,5-[3H]leucine, [1-14C]leucine and [1-14C]ketoisocaproate (ketoleucine) as tracers and on the basis of difference of amino acid levels in perfusion solution at the beginning and end of perfusion. In liver perfused with a solution of pH 7.2, we observed higher rates of proteolysis, protein synthesis, amino acid utilization and urea production. Furthermore, the liver perfused with a solution of pH 7.2 released a higher amount of proteins to perfusate than the liver perfused with a solution of pH 7.4. Enhanced decarboxylation of ketoisocaproate in liver perfused by a solution of a lower pH indicates increased catabolism of branched-chain amino acids (leucine, valine and isoleucine), decreased reamination of branched-chain keto acids to corresponding essential amino acids and increased ketogenesis from leucine.     

A specific binding site in Nb2 node lymphoma cells mediates the effects of didemnin B, an immunosuppressive cyclic peptide.

Didemnin B (DB) is a cyclic depsipeptide with a variety of biologic effects, including potent antiviral, antitumor, and immunosuppressive activities. Although its mechanism of action has been attributed to inhibition of DNA and protein synthesis, the exact cellular site of interaction has not been previously defined. Since DB is strongly antiproliferative in Nb2 node lymphoma cells, we investigated potential DB binding sites in these cells, using [3H]-DB (2.7 mCi/mg) as the radiolabeled ligand. Time course studies with Nb2 cells showed that steady state [3H]-DB binding was attained after 4 h. Scatchard analysis with resting cells yielded a Kd of 180 nM (200 ng/ml), and 7 x 10(6) binding sites/cell. The IC50 of DB inhibition of ongoing protein and DNA synthesis in Nb2 cells, measured 24 h after prolactin (PRL) stimulation, was also in the range of 100 ng/ml. Didemnin analogs, with alterations at critical amino acid residues, inhibited the synthesis of DNA and protein and competed with [3H]-DB binding with the same rank order of potency. This implies that this binding site may mediate the inhibition of macromolecule synthesis. Subcellular fractionation of [3H]-DB labeled Nb2 cells revealed that specific binding occurred predominantly in the 100,000 g cytosolic fraction. Comparison with cyclophilin and the FK506 binding protein, both cytosolic receptors, suggests that the DB binding site may also belong to the family of immunophilins.     

The synthesis of amylase in parotid glands of young and old rats

The age-related changes in the rate of synthesis of total and secretory proteins were examined in parotid glands of young (2 months) and old (24 months) rats. The differences in the rate of incorporation of radioactive leucine into acid-insoluble proteins of the gland indicate that the rate of protein synthesis declines with age in this gland. To determine whether the rate of synthesis of secretory proteins changes with age in this gland, the rates of incorporation of [3H]leucine into amylase, a major secretory protein of the gland, were compared by radioactivity determinations. For this comparison, amylase was precipitated with glycogen after incubating the gland slices in the presence of the labeled amino acid. The study shows that rate of synthesis of amylase declines significantly with age in this gland. The possible relationship between the decline in protein synthesis and the reduced level of secretory activity of the gland due to aging is discussed.     

Melatonin modulates diacylglycerol and arachidonic acid metabolism in the anterior pituitary of immature rats

In pituitary glands of immature rats prelabeled in vitro with [3H]arachidonic acid, melatonin diminished the luteinizing hormone-releasing hormone (LHRH)-induced increase in [3H]diacylglycerol accumulation as well as [3H]arachidonic acid release from the tissue. Melatonin reduced also LHRH-stimulated incorporation of [3H]glycerol into pituitary [3H]diacylglycerol. The effect was day-time dependent: in the evening experiment melatonin was effective at 0.1 nM concentration while in the morning it had no effect even at 10 nM concentration. The effect of melatonin was also abolished by pretreatment with pertussis toxin. Diacylglycerol and/or arachidonic acid might serve as 2nd messengers transducing the effect of melatonin at the cellular level.     

Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of lymphocyte proliferative responses by ribavirin.

When added to cultures of human peripheral blood lymphocytes, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibited antigen- and mitogen-induced proliferative responses as determined by [3H]thymidine incorporation. Dose-dependent suppressive effects were obtained when concentrations of 5 to 60 microgram of ribavirin per ml were added at culture initiation or up to 96 h thereafter. Ribavirin inhibited [3H]uridine and [3H]leucine incorporation by concanavalin A-activated and normal lymphocytes although not as severely as deoxyribonucleic acid synthesis. The capacity of ribavirin to interfere with lymphoproliferative responses was entirely reversed by guanosine and, to a lesser extent, by adenosine and xanthosine. These studies demonstrate that ribavirin is a reversible inhibitor of lymphocyte nucleic acid synthesis and suggest that the drug may be immunosuppressive when administered in vivo.     

Parasympathetic non-adrenergic, non-cholinergic-induced protein synthesis and mitogenic activity in rat parotid glands

Electrical stimulation of the parasympathetic auriculo-temporal nerve (40 Hz, 30 min), in the anaesthetized rat under - and -adrenoceptor blockade, increased [3H]thymidine and [3H]leucine uptake into the parotid glands by 80 and 263 %, respectively. The increase in response to parasympathetic stimulation was almost the same ([3H]thymidine 82 % and [3H]leucine 283 %) when atropine (2 mg kg-1 I.P. or I.V.) was included in the pretreatment. Neither intravenous infusion of vasoactive intestinal peptide (0.5-20 mg kg-1 min-1, 30 min) nor of bethanechol (10 mg kg-1 min-1, 30 min), under adrenoceptor blockade, increased the uptake of [3H]thymidine into the glands. However, these drugs increased [3H]leucine uptake, and in combination they interacted positively. Whereas vasoactive intestinal peptide is likely to be involved in the parasympathetic nerve-evoked protein synthesis, the nature of the non-adrenergic, non-cholinergic component(s) involved in the mitogenic response is presently unknown.     

The number of sodium ion pumping sites in skeletal muscle and its modification by insulin.

1. [3H]ouabain binding by frog sartorius muscles shows at least two components: one linked to inhibition of the pump and another not related to transport inhibition. This is suggested by the finding that [3H]ouabain uptake continued to increase when (a) the glycoside concentration was increased beyond that causing maximum transport inhibition, and (b) exposure times longer than those required to produce full inhibition were used. 2. A number of 1600 pumping sites per mum2 of membrane was estimated considering only the cylindrical surface of the muscle. 3. Insulin stimulated the ouabain-sensitive components of 22Na efflux and 134Cs influx. It also increased [3H]ouabain binding to a level of 1-7 times the total resting value. The increases in [3H]ouabain binding and in 22Na efflux followed a similar relationship with respect to insulin concentration. 4. Insulin stimulated the Na pump in muscles whose pumping sites had been inhibited by ouabain and then transferred to a glycoside-free solution. This stimulation was observed before detecting any recovery of the initial pumping activity. 5. When both the resting and the insulin-stimulated 22Na efflux had been blocked by ouabain, an additional dose of insulin, in a ouabain-free solution, had no further effects on 22Na efflux. 6. The effects of insulin were unaffected by cycloheximide or by high concentrations of butyryl derivatives of cyclic AMP and cyclic GMP. 7. We conclude that there are two pools of Na pumping sites in muscle cells: one active and another inactive. Insulin unmasks the inactive pumping sites by a mechanism that is independent of protein synthesis, increases in intracellular [Na] or decreases in intracellular [K].     

Effects of clofibrate administration to rats on their hepatocytes

Changes in the hepatocytes of rats treated with the hypolipidemic agent clofibrate for a 1 week period were investigated. During this time liver weight increased while, in contrast to peroxisomes, mitochondrial and microsomal protein were not changed significantly. While there was no effect on cytochrome oxidase activity carnitineacetyl transferase activity increased more than 20-fold. The urate oxidase activity of the homogenate decreased about 30% and palmitoyl-CoA oxidation increased more than 10 times. The ratio of protein to phospholipid in microsomes was not affected, nor were the rates of incorporation of [³H]leucine, [³H]glycerol and [³H]mevalonate into total protein, phospholipid and cholesterol, respectively. Injection of labeled glucosamine, mannose and galactose into the animals revealed an increased incorporation of these substances into lipid intermediates and endogenous luminal and membranous protein acceptors after clofibrate treatment. The significant stimulation of enzymes participating in hydroxylation reactions may be the main reason for the complex cellular changes brought about by clofibrate.     

Activation of endogenous type C virus by amino acid alcohols.

Amino acid alcohols were examined for their ability to inhibit protein synthesis and induce endogenous type C virus from Kirsten sarcoma virus (KiSV)-transformed Balb/c mouse cells. Histidinol and tyrosinol, amino alcohols of histidine and tyrosine, were very effective short-term activators of virus. Both inducers were very efficient inhibitors of protein synthesis reducing [³H]leucine incorporation by more than 90% in 1 hr. Two other alcohols, valinol and methioninol, also reduced protein synthesis but gave low activation. The activated virus had the host range of the xenotropic Balb virus:2, and following removal of inducer, the activated state decayed rapidly.