Measurement of hepatic protein synthesis in unrestrained mice-evaluation of the 'flooding technique'

Controversy exists regarding the validity of various techniques for estimating rates of protein synthesis in vivo. In the present report, we have compared estimates of hepatic protein synthesis in normal mice with a pulse labelling of [1-14C]leucine and calculated hepatic protein synthetic rates in a conventional two-pool model and in a five-pool compartment analysis. Results obtained with pulse labelling were also compared to those obtained in animals receiving a flooding dose of 1.5 mumol L-phenylalanine and 0.4 microCi [U-14C]phenylalanine per gram of body weight or 1.0 mumol L-leucine and 0.4 microCi [l-14C]leucine per gram of body weight. Estimates of protein synthesis were calculated with plasma free amino acid, liver acid-soluble fraction and acylated tRNA specific radioactivities as being representative of the precursor pool for protein synthesis. Rates of hepatic protein synthesis obtained with pulse labelling and either leu-tRNA or acid-soluble fractions of liver leucine as the precursor for protein synthesis gave similar results (37 +/- 5 vs 42 +/- 5% per day) in a two-pool model, but disagreed in a five-pool model (37 +/- 5 vs 6 +/- 2% per day). Estimates based on plasma enrichment in leucine were only one fifth of values obtained with tRNA in labelling experiments. When the plasma pool with tracer amino acids was used to indicate the precursor labelling of protein synthesis, values obtained with the flooding dose of either phenylalanine or leucine agreed with those obtained with pulse labelling and enrichment in tRNA (30 +/- 3 nmol min-1 vs 28 +/- 4 nmol min-1); with however no agreement when the enrichment in the liver mixed tissue pool was used (76 +/- 5 nmol min-1). Complete equilibration of the amino acid pools did not occur despite flooding. Therefore, the flooding technique may only represent an approximate method to measure protein synthesis in vivo, although it gives absolute values that agree well with results from labelling techniques based on tRNA enrichment provided the plasma pool is used as the precursor enrichment.     

Measurement of hepatic protein synthesis in unrestrained mice-evaluation of the ‘flooding technique’

Controversy exists regarding the validity of various techniques for estimating rates of protein synthesis in viva. In the present report, we have compared estimates of hepatic protein synthesis in normal mice with a pulse labelling of [1-¹⁴C]leucine and calculated hepatic protein synthetic rates in a conventional two-pool model and in a five-pool compartment analysis. Results obtained with pulse labelling were also compared to those obtained in animals receiving a flooding dose of 1.5 μmol L-phenylalanine and 0.4 μCi [U-¹⁴C]phenylalanine per gram of body weight or 1.0 μmol L-leucine and 0.4, μCi [I-¹⁴C]leucine per gram of body weight. Estimates of protein synthesis were calculated with plasma free amino acid, liver acid-soluble fraction and acylated tRNA specific radioactivities as being representative of the precursor pool for protein synthesis. Rates of hepatic protein synthesis obtained with pulse labelling and either leu-tRNA or acid-soluble fractions of liver leucine as the precursor for protein synthesis gave similar results (37 pL 5 vs 42 pL 5% per day) in a two-pool model, but disagreed in a fivepool model (37 pL 5 us 6 pL 2% per day). Estimates based on plasma enrichment in leucine were only one fifth of values obtained with tRNA in labelling experiments. When the plasma pool with tracer amino acids was used to indicate the precursor labelling of protein synthesis, values obtained with the flooding dose of either phenylalanine or leucine agreed with those obtained with pulse labelling and enrichment in tRNA (30 pL 3 nmol min-¹ us 28 pL 4 nmol min-¹); with however no agreement when the enrichment in the liver mixed tissue pool was used (76 pL 5 nmol min-¹). Complete equilibration of the amino acid pools did not occur despite flooding. Therefore, the flooding technique may only represent an approximate method to measure protein synthesis in vivo, although it gives absolute values that agree well with results from labelling techniques based on tRNA enrichment provided the plasma pool is used as the precursor enrichment.     

Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia

The present study was designed to assess the effects of dietary leucine supplementation on muscle protein synthesis and whole body protein kinetics in elderly individuals. Twenty healthy male subjects (70 ± 1 years) were studied before and after continuous ingestion of a complete balanced diet supplemented or not with leucine. A primed (3.6 μmol kg⁻¹) constant infusion (0.06 μmol kg⁻¹ min⁻¹) of l -[1-¹³C]phenylalanine was used to determine whole body phenylalanine kinetics as well as fractional synthesis rate (FSR) in the myofibrillar fraction of muscle proteins from vastus lateralis biopsies. Whole body protein kinetics were not affected by leucine supplementation. In contrast, muscle FSR, measured over the 5-h period of feeding, was significantly greater in the volunteers given the leucine-supplemented meals compared with the control group (0.083 ± 0.008 versus 0.053 ± 0.009% h⁻¹, respectively, P < 0.05). This effect was due only to increased leucine availability because only plasma free leucine concentration significantly differed between the control and leucine-supplemented groups. We conclude that leucine supplementation during feeding improves muscle protein synthesis in the elderly independently of an overall increase of other amino acids. Whether increasing leucine intake in old people may limit muscle protein loss during ageing remains to be determined.     

Protein metabolism in guanethidine-treated rats

The aim of the present study was to evaluate the effect of short-term adrenergic blockade on the rate of whole-body protein turnover and leucine oxidation, and on protein synthesis in specific tissues in male rats. Adrenergic blockade was induced by guanethidine (100 mg/kg body weight subcutaneously). The control group was treated with saline. On the second day, the parameters of whole-body protein and leucine metabolism were evaluated using a primed constant intravenous infusion of L-[1-(14)C]leucine. Protein synthesis in tissues was determined on the basis of L-[1-(14)C]leucine incorporation. Guanethidine treatment caused a decrease in norepinephrine in skeletal muscle. Whole-body leucine oxidation and leucine oxidized fraction were higher in guanethidine-treated rats. There was an insignificant effect of guanethidine on whole-body proteolysis, protein synthesis and leucine clearance. However, protein balance was negative due to the larger difference between protein synthesis and proteolysis in guanethidine-treated animals compared to controls. In guanethidine-treated rats, protein synthesis was higher in the gastrocnemius muscle and in the kidneys and lower in liver and spleen. Changes in the small intestine and colon were insignificant. In addition, a marked decrease in concentration of several amino acids has been observed in the liver, the kidneys and the spleen. It is concluded that adrenergic blockade induced by guanethidine is associated with significant changes in protein metabolism, leucine oxidation and amino acid concentrations in several tissues. The most important consequences of treatment are considered to be a negative effect on protein balance, increased protein turnover in skeletal muscle and kidneys and decreased protein synthesis in the liver and spleen. These changes may also be induced by administration of other sympathetic blocking agents, e.g. in treatment of hypertension.     

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.     

Incorporation of Leucine into Human Skeletal Muscle Proteins: A Study of Tissue Amino Acid Pools and their Role in Protein Biosynthesis

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-leucin had reached an equilibrium. The regression coefficients for the semilogarithmic plots of the labelling rate of proteins with 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.     

Recovery of Streptococcus mutans After Amino Acid Deprivation

The recovery of Streptococcus mutans FA-1 in a complete, chemically defined medium was examined after 1, 3, and 6 h of essential amino acid deprivation. Amino acids could be divided into two groups based on their effect on the relative rates of recovery: those amino acids (leucine and cystine) that are precursors of protein only, and amino acids (glutamate/glutamine or lysine) that are incorporated into both protein and cell wall peptidoglycan. Culture turbidity, deoxyribonucleic acid, ribonucleic acid, protein and cell wall peptidoglycan measurements indicated rapid recovery after leucine/cystine starvation periods. However, a 6-h leucine/cystine deprivation resulted in a slower exponential rate of growth (180-min doubling time compared to the normal doubling time of 85 to 90 min) after recovery. Glutamate/glutamine starvation, on the contrary, resulted in greatly extended recovery periods, especially after 3- and 6-h amino acid deprivations. Macromolecular synthesis was most severely affected by 6-h glutamate/glutamine starvation and required 6 to 10 h for recovery of an exponential rate. A delay in the recovery of deoxyribonucleic acid and cell wall peptidoglycan synthesis beyond that of the other macromolecules was observed after 1 and 3 h of deprivation with either leucine/cystine or glutamate/glutamine. However, after a 6-h amino acid deprivation, deoxyribonucleic acid synthesis recovered more rapidly than that of the other macromolecules studied. The results are discussed in terms of the nutritional environment of the oral cavity and its effect on the growth and survival of S. mutans.     

Age-related differences in the dose–response relationship of muscle protein synthesis to resistance exercise in young and old men

We investigated how myofibrillar protein synthesis (MPS) and muscle anabolic signalling were affected by resistance exercise at 20–90% of 1 repetition maximum (1 RM) in two groups (25 each) of post-absorptive, healthy, young (24 ± 6 years) and old (70 ± 5 years) men with identical body mass indices (24 ± 2 kg m⁻²). We hypothesized that, in response to exercise, anabolic signalling molecule phosphorylation and MPS would be modified in a dose-dependant fashion, but to a lesser extent in older men. Vastus lateralis muscle was sampled before, immediately after, and 1, 2 and 4 h post-exercise. MPS was measured by incorporation of [1,2-¹³C] leucine (gas chromatography–combustion–mass spectrometry using plasma [1,2-¹³C]α-ketoisocaparoate as surrogate precursor); the phosphorylation of p70 ribosomal S6 kinase (p70s6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was measured using Western analysis with anti-phosphoantibodies. In each group, there was a sigmoidal dose–response relationship between MPS at 1–2 h post-exercise and exercise intensity, which was blunted ( P < 0.05) in the older men. At all intensities, MPS fell in both groups to near-basal values by 2–4 h post-exercise. The phosphorylation of p70s6K and 4EBP1 at 60–90% 1 RM was blunted in older men. At 1 h post-exercise at 60–90% 1 RM, p70s6K phosphorylation predicted the rate of MPS at 1–2 h post-exercise in the young but not in the old. The results suggest that in the post-absorptive state: (i) MPS is dose dependant on intensity rising to a plateau at 60–90% 1 RM; (ii) older men show anabolic resistance of signalling and MPS to resistance exercise.     

Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-3C]leucine

Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasm [-13C]leucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.     

The effects of acute passive stretch on muscle protein synthesis in humans.

We examined the effect of an isolated bout of maximal tolerated passive stretch on fractional muscle protein synthetic rate in human soleus muscle. Eight healthy males performed two separate trials with the same leg: one session of passive stretch and one of intermittent active isometric contraction at a force equivalent to that which occurred during the passive stretch trial. This force was approximately 40% of maximum voluntary contraction force and produced volitional fatigue in approximately 27 min. Intermittent passive stretch, for the same duration, elicited a 6.1 degrees increase in joint angle (P<.0005) with silent electromyography. Fractional protein synthetic rate from experimental and control soleus in each trial was assessed from biopsy samples over the period 10-22 hr postexercise by the incorporation rate of L-[1-13C] leucine into muscle. Protein synthesis was elevated in the soleus of the exercised leg following the active contraction trial by 49% (P<.05) but not following the passive stretch trial. Results indicate that a single bout of maximal passive stretch does not significantly elevate fractional muscle protein synthetic rate in humans and thus suggests that muscle stretch per se is not the stimulus for the muscle hypertrophy that occurs with resistance training.     

Changes in RNA, DNA and protein content and the rates of protein synthesis and degradation during hypertrophy of the anterior latissimus dorsi muscle of the adult fowl (Gallus domesticus)

Hypertrophy of the anterior latissimus dorsi muscle of the adult fowl was induced by attaching a weight to one wing and after 2, 6 and 14 days the muscle wet weight, as well as protein, RNA and DNA contents were measured. After 6 days of hypertrophy the wet weight of the muscle had increased by 74%, the protein content by 44%, RNA by 203% and DNA by 83% compared with the contralateral control. Average rates of protein synthesis and degradation were measured over the 14 days of hypertrophy from the rate of loss of radioactivity in the muscle protein after pulse labelling using 3H-leucine. Results were compatible with a 50% increase in the protein synthesis rate with no detectable change in the degradation rate. The increase in synthesis rate was paralleled by a similar rise in the RNA concentration. The DNA content indicated that this increase in RNA resulted from an increased RNA production per nucleus as well as from an increased number of nuclei. The kinetics of the loss of total radioactivity from the muscle protein and the amount of free 3H-leucine remaining in the muscle free amino acid pool during hypertrophy indicated that reutilization of 3H-leucine was extensive. This was in spite of attempts aimed at minimizing reutilization by feeding fowls a high protein diet plus a daily supplement of unlabelled leucine.     

Effect of undernutrition on metabolic compartmentation of glutamate and on the incorporation of [14C] leucine into protein in the developing rat brain.

The effect of undernutrition on the rate of protein synthesis and the development of metabolic compartmentation of glutamate in the brain was investigated by using [U -14 C] leucine as precursor. In the brain of normal rats the incorporation rate of [14C] leucine into protein was at a maximum during the 3rd week after birth, but in the undernourished animal this rate was markedly lower. The biochemical maturation of the brain, followed in terms of the age-dependent increase in the glutamine/glutamate specific radioactivity ratio, was severly retarded in the undernourished animals, mainly as a result of a marked depression in the conversion of leucine carbon into glutamine. However these biochemical effects of undernutrition were reversible: on rehabilitation from Day 21-35 the rate of conversion of leucine carbon, both into proteins and glutamate and glutamine, was restored to normal.     

Effects of nitric oxide synthase inhibition by l-NAME on oxygen uptake kinetics in isolated canine muscle in situ

We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups ( n = 8 and 6) of healthy young men were studied over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload ( W _max). To label tissue proteins in muscle and tendon primed, constant infusions of [1-¹³C]leucine or [1-¹³C]valine and flooding doses of [¹⁵N] or [¹³C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography–mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h⁻¹), muscle collagen (0.054% h⁻¹), myofibrillar protein (0.121% h⁻¹), and sarcoplasmic protein (0.134% h⁻¹)). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation.     

Effects of liver ischemia on hepatic protein synthesis in vitro and in vivo

When an in vitro system is used to study the influence of ischemia on hepatic protein synthesis, an important question is whether alterations observed in vitro reflect changes in vivo. In the present study the effects of liver ischemia on protein synthesis were investigated in rats both in vitro and in vivo. Liver ischemia was induced by hepatic artery ligation. Protein synthesis in vitro was determined from leucine incorporation into proteins in liver slices incubated in a medium containing ¹⁴C-leucine (0.5 mmol/l) and in vivo from leucine incorporation into hepatic proteins after intraportal injection of a tracer dose of ¹⁴C-leucine. Leucine incorporation rate in non-ischemic liver was 0.16 pmol * g pror¹ h-¹ in vitro and 19.6 μmol g prot-¹. h^-1 in vivo. After hepatic artery ligation protein synthesis in vitro was reduced by about 60% and in vivo by about 80%. Thus, the relative changes were of the same magnitude in vitro and in vivo. This indicates that an in vitro system can be used to evaluate the effects of liver ischemia on hepatic protein synthesis.     

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.     

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.     

High triacylglycerol turnover rate in human skeletal muscle

In the present study we investigated the relationship between plasma fatty acids (FA) and intramuscular triacylglycerol (IMTAG) kinetics of healthy volunteers. With this aim [U-¹³C]-palmitate was infused for 10 h and FA kinetics determined across the leg. In addition, the rate of FA incorporation into IMTAG in vastus lateralis muscle was determined during two consecutive 4-h periods (2–6 h and 6–10 h). Fifty to sixty per cent of the FA taken up from the circulation were esterified into IMTAG, whereas 32 and 42% were oxidized between 2–6 and 6–10 h, respectively. IMTAG fractional synthesis rate was 3.4 ± 0.8% h⁻¹ and did not change between the two 4- h periods, despite an increase in arterial FA concentration  (34%, P < 0.01)  . IMTAG concentration was also unchanged, implying that the IMTAG fractional synthesis rate was balanced by an equal rate of breakdown. FA oxidation increased over time, which could be due to the observed decline in plasma insulin concentration  (−74%, P < 0.01)  . In conclusion, a substantial fraction of the fatty acids entering skeletal muscle in post-absorptive healthy individuals is esterified into IMTAG, due to its high turnover rate (29 h pool⁻¹). An increase in FA level, as a consequence of short-term fasting, does not seem to increase IMTAG synthesis rate and pool size.     

Acute effects of phenylbutyrate on glutamine,branched‐chain amino acid and protein metabolism in skeletal muscles of rats

Phenylbutyrate (PB) acts as chemical chaperone and histone deacetylase inhibitor, which is used to decrease ammonia in urea cycle disorders and has been investigated for use in the treatment of a number of lethal illnesses. We performed in vivo and in vitro experiments to examine the effects of PB on glutamine (GLN), branched‐chain amino acid (BCAA; valine, leucine and isoleucine) and protein metabolism in rats. In the first study, animals were sacrificed one hour after three injections of PB (300mg/kg b.w.) or saline. In the second study, soleus (SOL, slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles were incubated in a medium with or without PB (5 mM). L‐[1‐¹⁴C] leucine was used to estimate protein synthesis and leucine oxidation, and 3‐methylhistidine release was used to evaluate myofibrillar protein breakdown. PB treatment decreased GLN, BCAA and branched‐chain keto acids (BCKAs) in blood plasma, decreased BCAA and increased GLN concentrations in muscles, and increased GLN synthetase activities in muscles. Addition of PB to incubation medium increased leucine oxidation (55% in EDL, 29% in SOL), decreased BCKA and increased GLN in medium of both muscles, increased GLN in muscles, decreased protein synthesis in SOL and increased proteolysis in EDL. It is concluded that PB decreases BCAA, BCKA and GLN in blood plasma, activates BCAA catabolism and GLN synthesis in muscle and exerts adverse effects on protein metabolism. The results indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be a useful prospective agent which could be effective in management of maple syrup urine disease.     

The effect of unloading on protein synthesis in human skeletal muscle

Atrophy of skeletal muscle is observed in response to immobilization and lack of weight-bearing. The aim of this study was to investigate the effect of immobilization on muscle protein synthesis and associated biochemical parameters in skeletal muscle of healthy volunteers employing a standardized model of lower limb unloading. One leg was unloaded for 10 days, and percutaneous muscle biopsies were taken before and at the end of the unloading period. The capacity for protein synthesis, as reflected by the concentration of RNA, decreased by 16% (P < 0.05) although the fractional synthesis rate (FSR) of protein was not significantly changed after 10 days of unloading. Furthermore there was an increase in the concentration of the free branched chain amino acids in muscle by 48% (P < 0.05).     

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.